Methods of Improving Animal Performance

ABSTRACT

The present invention relates to methods of improving animal performance using animal feed comprising microbial polypeptides having lysozyme activity.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to methods of improving animal performanceusing animal feed comprising microbial polypeptides having lysozymeactivity.

Description of the Related Art

Lysozyme is an O-glycosyl hydrolase produced as a defensive mechanismagainst bacteria by many organisms. The enzyme causes the hydrolysis ofbacterial cell walls by cleaving the glycosidic bonds of peptidoglycan;an important structural molecule in bacteria. After having their cellwalls weakened by lysozyme action, bacterial cells lyse as a result ofumbalanced osmotic pressure.

Lysozyme naturally occurs in many organisms such as viruses, plants,insects, birds, reptiles and mammals. In mammals, Lysozyme has beenisolated from nasal secretions, saliva, tears, intestinal content, urineand milk. The enzyme cleaves the glycosidic bond between carbon number 1of N-acetylmuramic acid and carbon number 4 of N-acetyl-D-glucosamine.In vivo, these two carbohydrates are polymerized to form the cell wallpolysaccharide of many microorganisms.

Lysozyme has been classified into five different glycoside hydrolase(GH) families (CAZy, www.cazy.org): hen egg-white lysozyme (GH22), gooseegg-white lysozyme (GH23), bacteriophage T4 lysozyme (GH24),Sphingomonas flagellar protein (GH73) and Chalaropsis lysozymes (GH25).Lysozymes from the families GH23 and GH24 are primarily known frombacteriophages and have only recently been identified in fungi. Thelysozyme family GH25 has been found to be structurally unrelated to theother lysozyme families.

Lysozyme has traditionally been extracted from hen egg white due to itsnatural abundance and until very recently hen egg white lysozyme was theonly lysozyme investigated for use in animal feed. Lysozyme extractedfrom hen egg white is the primary product available on the commercialmarket, but does not cleave N,6-O-diacetylmuramic acid in e.g.Staphylococcus aureus cell walls and is thus unable to lyse thisimportant human pathogen among others (Masschalck B, Deckers D, MichielsC W (2002), “Lytic and nonlytic mechanism of inactivation ofgram-positive bacteria by lysozyme under atmospheric and highhydrostatic pressure”, J Food Prot. 65(12):1916-23).

WO2000/21381 discloses a composition comprising at least twoantimicrobial enzymes and a polyunsaturated fatty acid, wherein one ofthe antimicrobial enzymes was a GH22 lysozyme from chicken egg white.GB2379166 discloses a composition comprising a compound that disruptsthe peptidoglycan layer of bacteria and a compound that disrupts thephospholipid layer of bacteria, wherein the peptidoglycan disruptingcompound was a GH22 lysozyme from chicken egg white.

WO2004/026334 discloses an antimicrobial composition for suppressing thegrowth of enteric pathogens in the gut of livestock comprising (a) acell wall lysing substance or its salt, (b) a antimicrobial substance,(c) a sequestering agent and (d) a lantibiotic, wherein the cell walllysing substance or its salt is a GH22 lysozyme from hen egg white.

The growing world population and increasing demand on animal proteinreinforces the relevance of improving the growth performance of animals.Thus solutions which improve growth rate using less animal feed orresulting in bigger animals using the same amount of feed is always ofinterest to farmers. The object of this invention is to provide anothersolution to this global issue.

SUMMARY OF THE INVENTION

The invention relates to a method of improving the European ProductionEfficiency Factor (EPEF) and/or feed conversion ratio (FCR) of amonogastric animal comprising administering an animal feed or animal feeadditive comprising one or more microbial lysozymes to the monogastricanimal wherein the microbial lysozyme obtained or obtainable from thekingdom Fungi and is administered at a level of 8 to 250 ppm enzymeprotein per kg animal feed.

The invention further relates to a method of increasing the proportionof bacteria of genus Faecalibacterium in the microbiome of the GI tractof a monogastric animal comprising administering to the animal an animalfeed or animal feed additive comprising one or more microbial lysozymesadministered at a level of 8 to 250 ppm enzyme protein per kg animalfeed.

OVERVIEW OF SEQUENCE LISTING

SEQ ID NO: 1 is the mature amino acid sequence of a wild type GH25lysozyme from Acremonium alcalophilum with N-terminal SPIRR as describedin WO 2013/076253.

SEQ ID NO: 2 is the gene sequence of the GH24 lysozyme as isolated fromTrichophaea saccata.

SEQ ID NO: 3 is the amino acid sequence as deduced from SEQ ID NO: 2.

SEQ ID NO: 4 is the mature amino acid sequence of a wild type GH24lysozyme from Trichophaea saccata.

SEQ ID NO: 5 is the mature amino acid sequence of a wild type GH22lysozyme from Gallus gallus (hen egg white lysozyme).

SEQ ID NO: 6 is primer F-80470.

SEQ ID NO: 7 is primer R-80470.

SEQ ID NO: 8 is primer 8643.

SEQ ID NO: 9 is primer 8654.

SEQ ID NO: 10 is the forward primer 27F.

SEQ ID NO: 11 is the reverse primer 534R.

SEQ ID NO: 12 is the mature amino acid sequence of a wild type GH25lysozyme from Acremonium alcalophilum as described in WO 2013/076253.

SEQ ID NO: 13 is the sequence representing the V1-V3 region of the 16SrRNA gene in OTU_20 classified as the bacterial genus Faecalibacteriumfrom in vivo trial 4 (Example 11).

SEQ ID NO: 14 is the sequence representing the V1-V3 region of the 16SrRNA gene in OTU_27 classified as the bacterial genus Faecalibacteriumfrom in vivo trial 4 (Example 11).

SEQ ID NO: 15 is the sequence representing the V1-V3 region of the 16SrRNA gene in OTU_85 classified as the bacterial genus Faecalibacteriumfrom in vivo trial 4 (Example 11).

FIGURES

FIG. 1: Principal component analysis plot showing a shift in themicrobial composition in the chicken gut upon treatment with thelysozyme of SEQ ID NO: 1 at 50 ppm from in vivo broiler trial 4.

FIG. 2: Boxplot of observed changes in the composition of the chickengut microbiota for in vivo broiler trial 4 (SEQ ID NO: 1 at 50 ppm) atOTU/species level. The boxplot was generated with the R programminglanguage.

FIG. 3: Boxplot of observed changes in the composition of the chickengut microbiota for in vivo broiler trial 4 (SEQ ID NO: 1 at 50 ppm) atgenus level. The boxplot was generated with the R programming language.

FIG. 4: Boxplot of observed changes in the composition of the chickengut microbiota for in vivo broiler trial 4 (SEQ ID NO: 1 at 50 ppm) atorder level. The boxplot was generated with the R programming language.

DEFINITIONS

Animal feed: The term “animal feed” refers to any compound, preparation,or mixture suitable for, or intended for intake by an animal. Animalfeed for a monogastric animal typically comprises concentrates as wellas vitamins, minerals, enzymes, direct fed microbial, amino acids and/orother feed ingredients (such as in a premix) whereas animal feed forruminants generally comprises forage (including roughage and silage) andmay further comprise concentrates as well as vitamins, minerals, enzymesdirect fed microbial, amino acid and/or other feed ingredients (such asin a premix).

Antimicrobial activity: The term “antimicrobial activity” is definedherein as an activity that kills or inhibits the growth ofmicroorganisms, such as, algae, archea, bacteria, fungi and/orprotozoans. The antimicrobial activity can, for example, be bactericidalmeaning the killing of bacteria or bacteriostatic meaning the preventionof bacterial growth. The antimicrobial activity can include catalyzingthe hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in a peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrins. Antimicrobial activitycan also include the lysozyme binding to the surface of themicroorganism and inhibiting its growth. The antimicrobial effect canalso include the use of the lysozymes of the present invention foractivation of bacterial autolysins, as an immunostimulator, byinhibiting or reducing bacterial toxins and by an opsonin effect.

For the purpose of the present invention, antimicrobial activity isdetermined according to the antimicrobial assay described in Example 6(“Determination of antimicrobial activity”). Antimicrobial activity isdetermined if there is a clearning zone when using 50% Mueller-Hintonbroth, pH 6. Preferably the diameter of the clearing zone is 4 mm ormore.

Concentrates: The term “concentrates” means feed with high protein andenergy concentrations, such as fish meal, molasses, oligosaccharides,sorghum, seeds and grains (either whole or prepared by crushing,milling, etc. from e.g. corn, oats, rye, barley, wheat), oilseed presscake (e.g. from cottonseed, safflower, sunflower, soybean (such assoybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake,yeast derived material and distillers grains (such as wet distillersgrains (WDS) and dried distillers grains with solubles (DDGS)).

European Production Efficiency Factor (EPEF): The European ProductionEfficiency Factor is a way of comparing the performance of animals. Thissingle-figure facilitates comparison of performance within and amongfarms and can be used to assess environmental, climatic and animalmanagement variables. The EPEF is calculated as [(liveability(%)×Liveweight (kg))/(Age at depletion (days)×FCR)]×100, whereinlivability is the percentage of animals alive at slaughter, Liveweightis the average weight of the animals at slaughter, age of depletion isthe age of the animals at slaughter and FCR is the feed conversion ratioat slaughter.

Faecalibacterium: It is known (Vĕtrovský T, Baldrian P (2013) TheVariability of the 16S rRNA Gene in Bacterial Genomes and ItsConsequences for Bacterial Community Analyses. PLoS ONE 8(2): e57923.doi: 10.1371/journal.pone.0057923) that the 16S rRNA gene sequenceidentity varies within a genus. It has been shown that the mean identityis 95.56 with a standard deviation of 3.68. It was also found that 12.2%of genera contain species with mean pairwise 16S rRNA gene similaritybelow 90%.

SEQ ID NO: 13 to 15 inclusive contains 16S rRNA gene sequencesclassified as genus Faecalibacterium from in vivo trial 4 (Example 11)where the V1-V3 region of the 16S rRNA gene was used for amplification.The classification was performed using the program “rdp classifier”v.2.2. OTU_20 (SEQ ID NO: 13) from in vivo trial 4 (Example 1) was themost abundant Faecalibacterium in this trial.

Thus strains are hereby defined as Faecalibacterium wherein the sequenceidentity of the V1-V3 region of the 16S rRNA gene of said strain has atleast 90% e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to one or more of SEQ ID NO: 13 to 15. Preferablythe sequence identity of the V1-V3 region of the 16S rRNA gene of saidstrain has at least 90%, more preferably at least 93%, even morepreferably at least 95%, even more preferably at least 96%, even morepreferably at least 97%, even more preferably at least 98%, even morepreferably at least 99%, or most preferably 100% sequence identity toSEQ ID NO: 13.

Feed Conversion Ratio (FCR): FCR is a measure of an animal's efficiencyin converting feed mass into increases of the desired output. Animalsraised for meat—such as swine, poultry and fish—the output is the massgained by the animal. Specifically FCR is calculated as feed intakedivided by weight gain, all over a specified period. Improvement in FCRmeans reduction of the FCR value. A FCR improvement of 2% means that theFCR was reduced by 2%.

Forage: The term “forage” as defined herein also includes roughage.Forage is fresh plant material such as hay and silage from forageplants, grass and other forage plants, seaweed, sprouted grains andlegumes, or any combination thereof. Examples of forage plants areAlfalfa (lucerne), birdsfoot trefoil, brassica (e.g. kale, rapeseed(canola), rutabaga (swede), turnip), clover (e.g. alsike clover, redclover, subterranean clover, white clover), grass (e.g. Bermuda grass,brome, false oat grass, fescue, heath grass, meadow grasses, orchardgrass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats,rye, sorghum, soybeans and wheat and vegetables such as beets. Foragefurther includes crop residues from grain production (such as cornstover; straw from wheat, barley, oat, rye and other grains); residuesfrom vegetables like beet tops; residues from oilseed production likestems and leaves form soy beans, rapeseed and other legumes; andfractions from the refining of grains for animal or human consumption orfrom fuel production or other industries.

Fragment: The term “fragment” means a polypeptide or a catalytic domainhaving one or more (e.g., several) amino acids absent from the aminoand/or carboxyl terminus of a mature polypeptide or domain; wherein thefragment has lysozyme. In one aspect, a fragment comprises at least 170amino acids, such as at least 175 amino acids, at least 177 amino acids,at least 180 amino acids, at least 185 amino acids, at least 190 aminoacids, at least 195 amino acids or at least 200 amino acids of SEQ IDNO: 1 and has lysozyme activity.

In another aspect, a fragment comprises at least 210 amino acids, suchas at least 215 amino acids, at least 220 amino acids, at least 225amino acids, at least 230 amino acids, at least 235 amino acids or atleast 240 amino acids of SEQ ID NO: 4 and has lysozyme activity.

In one aspect, a fragment comprises at least 170 amino acids, such as atleast 175 amino acids, at least 177 amino acids, at least 180 aminoacids, at least 185 amino acids, at least 190 amino acids, at least 195amino acids or at least 200 amino acids of SEQ ID NO: 12 and haslysozyme activity.

Increases the proportion of bacteria of x in the microbiota of the GItract of an animal: The term “increases the proportion of bacteria of xin the microbiota of the GI tract of an animal” means that the quantityof bacteria of a specific taxonomic rank (e.g. order or genus) hasincreased compared to a control sample. Samples of animal microbiota canbe taken from the gut (i.e. gastrointestinal tract) of an animal (e.g.from broiler ceca or from the colon or ileum of swine) and analysed byexamining the sequences (reads) of the 16S rRNA genes in the sample. Thereads of the 16S rRNA genes can be clustered together based on sequenceidentity and each cluster can be compared to a database of knownsequences of the 16S rRNA gene to identify the type of bacteria in thatcluster. The clusters can be merged at different taxonomic levels(phylum, class, order, family, genus or species) to give a quantativeanalysis of the amount of bacteria within each taxonomy level over theentire sample

By comparing the clusters from a control animal to an animaladministered with a lysozyme of the invention, differences in themicrobiota can be determined. Thus in one example, the proportion ofbacteria of genus Faecalibacterium in the microbiota taken from broilersadministered with a lysozyme of the invention increased from 0.22% to3.67% (see table 9.2) compared to control (i.e. broilers notadministered with a lysozyme). Thus in this example the proportion ofbacteria of genus Faecalibacterium increased by 3.45%, which correspondsto an increase by a factor of 16.53.

In another example, the proportion of bacteria of order Clostridiales inthe microbiota taken from broilers administered with a lysozyme of theinvention increased from 32.1% to 37.6% (see table 9.4) compared tocontrol. Thus in this example the proportion of bacteria of orderClostridiales decreased by 5.3%, which corresponds to an increase by afactor of 1.17.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Lysozyme activity: The term “lysozyme activity” means the enzymatichydrolysis of the 1,4-beta-linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in a peptidoglycan or betweenN-acetyl-D-glucosamine residues in chitodextrins, resulting inbacteriolysis due to osmotic pressure. Lysozyme belongs to the enzymeclass EC 3.2.1.17. Lysozyme activity is typically measured byturbidimetric determination. The method is based on the changes inturbidity of a suspension of Micrococcus luteus ATCC 4698 induced by thelytic action of lysozyme. In appropriate experimental conditions thesechanges are proportional to the amount of lysozyme in the medium (c.f.INS 1105 of the Combined Compendium of Food Additive Specifications ofthe Food and Agriculture Organisation of the UN (www.fao.org)). For thepurpose of the present invention, lysozyme activity is determinedaccording to the turbidity assay described in example 5 (“Determinationof Lysozyme Activity”). In one aspect, the polypeptides of the presentinvention have at least 20%, e.g., at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least100% of the lysozyme activity of SEQ ID NO: 1. In one aspect, thepolypeptides of the present invention have at least 20%, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or at least 100% of the lysozyme activity of SEQ IDNO: 4. In one aspect, the polypeptides of the present invention have atleast 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, or at least 100% of thelysozyme activity of SEQ ID NO: 12.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc.

Microbial lysozyme: The term “microbial lysozyme” means a polypeptidehaving lysozyme activity which is obtained or obtainable from amicrobial source. Examples of microbial sources are fungi; i.e. thelysozyme is obtained or obtainable from the kingdom Fungi, wherein theterm kingdom is the taxonomic rank. In particular, the microbiallysozyme is obtained or obtainable from the phylum Ascomycota, such asthe sub-phylum Pezizomycotina, wherein the terms phylum and sub-phylumis the taxonomic ranks.

If the taxonomic rank of a polypeptide is not known, it can easily bedetermined by a person skilled in the art by performing a BLASTP searchof the polypeptide (using e.g. the National Center for BiotechnologyInformation (NCIB) website http://www.ncbi.nlm.nih.gov/) and comparingit to the closest homologues. An unknown polypeptide which is a fragmentof a known polypeptide is considered to be of the same taxonomicspecies. An unknown natural polypeptide or artificial variant whichcomprises a substitution, deletion and/or insertion in up to 10positions is considered to be from the same taxonomic species as theknown polypeptide.

Monogastric animal: The term “monogastric animal” refers to any animalwhich has a simple single-chambered stomach except humans. Examples ofmonogastric animals include pigs or swine (including, but not limitedto, piglets, growing pigs, and sows); poultry such as turkeys, ducks,quail, guinea fowl, geese, pigeons (including squabs) and chicken(including but not limited to broiler chickens (referred to herein asbroiles), chicks, layer hens (referred to herein as layers)); horses(including but not limited to hotbloods, coldbloods and warm bloods)crustaceans (including but not limited to shrimps and prawns) and fish(including but not limited to amberjack, arapaima, barb, bass, bluefish,bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char,cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish,gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel,milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch,pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner,sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish,sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleyeand whitefish).

Operational taxonomic unit (OTU): The term “Operational taxonomic unit”means a cluster of sequences with a certain degree of similarity. Inthis case, 97 percent is chosen as the threshold for assigning sequencesof the 16S rRNA gene to different OTUs, meaning that all sequenceswithin a single OTU have at least 97 percent sequence identity. At thisidentity level each OTU is often considered (or assumed) to represent asingle bacterial species.

Roughage: The term “roughage” means dry plant material with high levelsof fiber, such as fiber, bran, husks from seeds and grains and cropresidues (such as stover, copra, straw, chaff, sugar beet waste).

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Substantially pure polypeptide: The term “substantially purepolypeptide” means a preparation that contains at most 10%, at most 8%,at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%,and at most 0.5% by weight of other polypeptide material with which itis natively or recombinantly associated. Preferably, the polypeptide isat least 92% pure, e.g., at least 94% pure, at least 95% pure, at least96% pure, at least 97% pure, at least 98% pure, at least 99%, at least99.5% pure, and 100% pure by weight of the total polypeptide materialpresent in the preparation. The polypeptides of the present inventionare preferably in a substantially pure form. This can be accomplished,for example, by preparing the polypeptide by well known recombinantmethods or by classical purification methods.

Variant: The term “variant” means a polypeptide having lysozyme activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, of one or more (several) amino acid residues at one or more(e.g., several) positions. A substitution means replacement of the aminoacid occupying a position with a different amino acid; a deletion meansremoval of the amino acid occupying a position; and an insertion meansadding 1, 2, or 3 amino acids adjacent to and immediately following theamino acid occupying the position.

In one aspect, a lysozyme variant according to the invention maycomprise from 1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from 1to 25; from 1 to 30; from 1 to 35; from 1 to 40; from 1 to 45; or from1-50, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 alterationsand have at least 20%, e.g., at least 40%, at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%of the lysozyme activity of the parent lysozyme, such as SEQ ID NO: 1,SEQ ID NO: 4 or SEQ ID NO: 12.

DETAILED DESCRIPTION OF THE INVENTION Methods of Improving AnimalPerformance

It has been surprisingly found that supplementing an animal feed with amicrobial lysozyme results in a significant performance benefit inmonogastric animals, such as broilers and piglets, compared to an animalfeed without the microbial lysozyme. This is surprising since improvedanimal performance using a microbial lysozyme has never previously beendemonstrated.

It has furthermore been discovered that the microbiota of the GI tractof an animal, such as broilers, is significantly altered byadministering a lysozyme of the invention. In one of the in vivo broilertrials, samples from the broiler ceca were taken for microbial community(microbiome) analysis and it was surprisingly discovered that:

-   -   (a) treatment with a GH25 lysozyme (SEQ ID NO: 1) leads to a        higher proportion of a bacterial species of the genus        Faecalibacterium in the chicken gut and this shift is associated        with increased European Production Efficiency Factor (EPEF) in        chickens and this bacterial species has 96% identity to the        species Faecalibacterium prausnitzii;    -   (b) treatment with a GH25 lysozyme (SEQ ID NO: 1) leads to a        higher proportion of bacteria of the genus Faecalibacterium in        the chicken gut and this shift is associated with increased        European Production Efficiency Factor (EPEF) in chickens;    -   (c) treatment with a GH25 lysozyme (SEQ ID NO: 1) leads to a        higher proportion of bacteria of the order Clostridiales in the        chicken gut and this shift is associated with increased European        Production Efficiency Factor (EPEF) in chickens;    -   (d) treatment with a GH25 lysozyme (SEQ ID NO: 1) leads to a        lower proportion of bacteria of the order Bacteroidales in the        chicken gut and this shift is associated with increased European        Production Efficiency Factor (EPEF) in chickens.

Treatment of chickens with a GH25 lysozyme (SEQ ID NO: 1) resulted inhigher levels of bacteria within the genus Faecalibacterium in thechicken gut environment. The closest known species is Faecalibacteriumprausnitzii, which is an obligate anaerobe rod-shapedbutyrate producingmicroorganism belonging to the phylum Firmicutes (Duncan et al. 2002,Int J Syst Evol Microbiol 52(Pt 6):2141-2146). It is abundant in thefeces of several animal species (Haenen D, et al. “A diet high inresistant starch modulates microbiota composition, SOFA concentrations,and gene expression in pig intestine”, J Nutr. 2013; 143: 274-283.; LundM, Bjerrum L, Pedersen K. “Quantification of Faecalibacteriumprausnitzii- and Subdoligranulum variable-like bacteria in the cecum ofchickens by real-time PCR”, Poult Sci. 2010; 89: 1217-1224). In humans,high levels of F. prausnitzii have been associated with obesity(Balamurugan R, et al “Quantitative differences in intestinalFaecalibacterium prausnitzii in obese Indian children”, Br J Nutr. 2010;103: 335-338), while a low abundance of F. prausnitzii has been linkedto Inflammatory Bowel Disease (IBD, i.e. Crohn's disease (Sokol H, etal. “Faecalibacterium prausnitzii is an anti-inflammatory commensalbacterium identified by gut microbiota analysis of Crohn diseasepatients”, Proc Natl Acad Sci USA. 2008; 105: 16731-16736) andulcerative colitis (Machiels K, et al. “A decrease of thebutyrate-producing species Roseburia hominis and Faecalibacteriumprausnitzii defines dysbiosis in patients with ulcerative colitis”, Gut.2013. doi: 10.1136/gutjnl-2013-304833)).

Additionally, the butyrate produced by F. prausnitzii is both an energysource to enterocytes and act as an anti-inflammatory agent (Miguel S,et al. “Identification of metabolic signatures linked toanti-inflammatory effects of Faecalibacterium prausnitzii”, MBio. 2015;6:doi: 10.1128/mBio.00300-15bioinf). Thus without wishing to be bound bytheory, it is believed that the GH25 lysozyme of the invention increasethe proportion of butyrate producing bacteria (such as those from theorder Clostridiales and specifically the genus Faecalibacterium).

Thus the invention relates to a method of improving the EuropeanProduction Efficiency Factor (EPEF) and/or feed conversion ratio (FCR)of a monogastric animal comprising administering an animal feed oranimal feed additive comprising one or more microbial lysozymes to themonogastric animal, wherein the microbial lysozyme is administered at alevel of 8 to 250 ppm enzyme protein per kg animal feed.

In a preferred embodiment, the improvement is compared to an animal feedor animal feed additive wherein the microbial lysozyme is not present(herein referred to as the negative control).

In one embodiment, the EPEF is improved by at least 1%, such as by atleast 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least 3.5%, atleast 4% or at least 5% compared to the control. In another embodiment,the EPEF is improved by between 1% and 15%, such as between 1% and 12%,between 1% and 10%, 1.5% and 8%, 2.0% and 7% compared to the control, orany combination of these intervals.

In one embodiment, the FCR is improved by at least 1%, such as by atleast 1.25%, at least 1.5%, at least 1.75% or at least 2.0% compared tothe control. In another embodiment, the FCR is improved by between 1%and 5%, such as between 1% and 4%, between 1% and 3%, 1.25% and 2.5%,1.5% and 2% compared to the control, or any combination of theseintervals.

In one embodiment, the microbial lysozyme is dosed at a level of 9 to200 ppm enzyme protein per kg animal feed, such as 10 to 150 ppm, 11 to125 ppm, 12 to 100 ppm, 13 to 75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25to 75 ppm or 30 to 60 ppm enzyme protein per kg animal feed, or anycombination of these intervals.

In one embodiment, the monogastric animal is a selected from the groupconsisting of swine, piglet, growing pig, sow, poultry, turkey, duck,quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer,pullet and chick, horse, crustaceans, shrimps, prawns, fish, amberjack,arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama,carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie,dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut,java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet,paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa,sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook,sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia,trout, tuna, turbot, vendace, walleye and whitefish. In a preferredembodiment, the monogastric animal is a selected from the groupconsisting of swine, piglet, growing pig, sow, poultry, turkey, duck,quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer,pullet and chick. In a more preferred embodiment, the monogastric animalis a selected from the group consisting of swine, piglet, growing pig,sow, chicken, broiler, layer, and chick.

In one embodiment, the microbial lysozyme has antimicrobial activitytowards Clostridium perfringens. In an embodiment, the microbiallysozyme has at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 100% of the antimicrobial activityof SEQ ID NO: 1 against Clostridium perfringens under the conditions 50%MHB, pH 6. In an embodiment, the microbial lysozyme has at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 100% of the antimicrobial activity of SEQ ID NO: 4 againstClostridium perfringens under the conditions 50% MHB, pH 6. In anembodiment, the microbial lysozyme has at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95% or at least 100% ofthe antimicrobial activity of SEQ ID NO: 12 against Clostridiumperfringens under the conditions 50% MHB, pH 6. Antimicrobial activitytowards Clostridium perfringens can be determined according to theantimicrobial assay described in Example 6.

In another embodiment, the invention relates to a composition comprisinga microbial lysozyme for the treatment of Clostridium perfringens in amonogastric animal wherein the composition improves the EuropeanProduction Efficiency Factor (EPEF) and/or feed conversion ratio (FCR)of the monogastric animal.

In one embodiment, the microbial lysozyme is fed to the animal frombirth until slaughter. In a preferred embodiment the microbial lysozymeis fed to the animal on a daily basis from birth until slaughter. Inanother preferred embodiment the microbial lysozyme is fed to the animalon a daily basis for at least 10 days, such as at least 15 days or atleast 20 days (where the days can be continuous or non-continuous)during the life span of the animal. In embodiment, the microbiallysozyme is fed to the animal for 10-20 days followed by a non-treatmentperiod of 5-10 days, and this cycle is repeated during the life span ofthe animal.

In a further embodiment, the microbial lysozyme is fed to broilers forthe first 49 days after hatching. In a further embodiment, the microbiallysozyme is fed to broilers for the first 36 days after hatching. In afurther embodiment, the microbial lysozyme is fed to broilers on days 22to 36 after hatching. In a further embodiment, the microbial lysozyme isfed to broilers during the pre-starter (days 1-7) period. In a furtherembodiment, the microbial lysozyme is fed to broilers during the starter(days 8-22) period. In a further embodiment, the microbial lysozyme isfed to broilers during the pre-starter (days 1-7) and starter (days8-22) period.

In a further embodiment, the microbial lysozyme is fed to layers duringthe life span of the animal. In a further embodiment, the microbiallysozyme is fed to layers for 76 weeks from hatching. In a furtherembodiment, the microbial lysozyme is fed to layers during the layingperiod, (from ca. week 18). In a further embodiment, the microbiallysozyme is fed to layers during the laying period but withheld duringthe forced molting period.

In a further embodiment, the microbial lysozyme is fed to turkeys duringlife span of the animal. In a further embodiment, the microbial lysozymeis fed to turkeys for 24 weeks from hatching. In a further embodiment,the microbial lysozyme is fed to turkeys for the first 16 weeks fromhatching (for hens) and for the first 20 weeks for hatching (for toms).

In a further embodiment, the microbial lysozyme is fed to swine duringlife span of the animal. In a further embodiment, the microbial lysozymeis fed to swine for 27 weeks from birth. In a further embodiment, themicrobial lysozyme is fed to piglets from birth to weaning (at 4 weeks).In a further embodiment, the microbial lysozyme is fed to piglets forthe first 6 weeks from birth (4 weeks of lactation and 2 weekspost-weaning). In a further embodiment, the microbial lysozyme is fed toweaning piglets during the pre-starter (days 1-14 after weaning). In afurther embodiment, the microbial lysozyme is fed to weaning pigletsduring the starter (days 15-42 after weaning) period. In a furtherembodiment, the microbial lysozyme is fed to weaning piglets during thepre-starter (days 1-14 after weaning) and starter (days 15-42 afterweaning) period. In a further embodiment, the microbial lysozyme is fedto swine during the grower/fattening period (week 10 to ca. week 27after birth).

In one embodiment, the microbial lysozyme is of fungal origin. In anembodiment, the microbial lysozyme is obtained or obtainable from thephylum Ascomycota, such as the sub-phylum Pezizomycotina.

In one embodiment, the microbial lysozyme comprises one or more domainsselected from the list consisting of GH24 and GH25.

In one embodiment, the method further increases the proportion ofbacteria of genus Faecalibacterium in the microbiota of the GI tract ofan animal. In an embodiment, the proportion of bacteria of genusFaecalibacterium is increased by at least 1%, such as at least 2%, atleast 3%, at least 4% or at least 5%. In an alternative embodiment, theproportion of bacteria of genus Faecalibacterium is increased by afactor of at least 1.25, such as at least 1.50, at least 1.75, at least2.0, at least 2.5 or at least 3.0.

In one embodiment, the method improves the European ProductionEfficiency Factor (EPEF) of an animal by at least 1% and increases theproportion of bacteria of genus Faecalibacterium in the microbiota ofthe GI tract of an animal. In an embodiment, the proportion of bacteriaof genus Faecalibacterium is increased by at least 1%, such as at least2%, at least 3%, at least 4% or at least 5% and the EPEF is increased byat least 1.25%, preferably by at least 1.5%, at least 1.75%, at least2%, at least 3%, at least 4% or most preferably by at least 5%.

In one embodiment, the method improves the European ProductionEfficiency Factor (EPEF) of an animal by at least 1% and increases theproportion of bacteria of genus Faecalibacterium in the microbiota ofthe GI tract of an animal. In an embodiment, the proportion of bacteriaof genus Faecalibacterium is increased by factor of at least 1.25, suchas at least 1.50, at least 1.75, at least 2.0, at least 2.5 or at least3.0 and the EPEF is increased by at least 1.25%, preferably by at least1.5%, at least 1.75%, at least 2%, at least 3%, at least 4% or mostpreferably by at least 5%.

In one embodiment, the method improves the Feed Conversion Ratio (FCR)of an animal by at least 1% and increases the proportion of bacteria ofgenus Faecalibacterium in the microbiota of the GI tract of an animal.In an embodiment, the proportion of bacteria of genus Faecalibacteriumis increased by at least 1%, such as at least 2%, at least 3%, at least4% or at least 5% and the FCR is increased by at least 1.25%, preferablyby at least 1.5% or most preferably by at least 1.75%.

In one embodiment, the method improves the Feed Conversion Ratio (FCR)of an animal by at least 1% and increases the proportion of bacteria ofgenus Faecalibacterium in the microbiota of the GI tract of an animal.In an embodiment, the proportion of bacteria of genus Faecalibacteriumis increased by factor of at least 1.25, such as at least 1.50, at least1.75, at least 2.0, at least 2.5 or at least 3.0 and the FCR isincreased by at least 1.25%, preferably by at least 1.5% or mostpreferably by at least 1.75%.

In one embodiment, the method further increases the proportion ofbacteria of genus Faecalibacterium in the microbiota of the GI tract ofan animal, wherein the bacteria of genus Faecalibacterium comprise 16SrRNA that has at least 90% e.g., at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13. In anembodiment, the proportion of bacteria of genus Faecalibacterium isincreased by at least 1%, such as at least 2%, at least 3%, at least 4%or at least 5%. In an alternative embodiment, the proportion of bacteriaof genus Faecalibacterium is increased by a factor of at least 1.25,such as at least 1.50, at least 1.75, at least 2.0, at least 2.5 or atleast 3.0.

In one embodiment, the method improves the European ProductionEfficiency Factor (EPEF) of an animal by at least 1% and increases theproportion of bacteria of genus Faecalibacterium in the microbiota ofthe GI tract of an animal, wherein the bacteria of genusFaecalibacterium comprise 16S rRNA that has at least 90% e.g., at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto SEQ ID NO: 13. In an embodiment, the proportion of bacteria of genusFaecalibacterium is increased by at least 1%, such as at least 2%, atleast 3%, at least 4% or at least 5% and the EPEF is increased by atleast 1.25%, preferably by at least 1.5%, at least 1.75%, at least 2%,at least 3%, at least 4% or most preferably by at least 5%.

In one embodiment, the method improves the European ProductionEfficiency Factor (EPEF) of an animal by at least 1% and increases theproportion of bacteria of genus Faecalibacterium in the microbiota ofthe GI tract of an animal, wherein the bacteria of genusFaecalibacterium comprise 16S rRNA that has at least 90% e.g., at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto SEQ ID NO: 13. In an embodiment, the proportion of bacteria of genusFaecalibacterium is increased by factor of at least 1.25, such as atleast 1.50, at least 1.75, at least 2.0, at least 2.5 or at least 3.0and the EPEF is increased by at least 1.25%, preferably by at least1.5%, at least 1.75%, at least 2%, at least 3%, at least 4% or mostpreferably by at least 5%.

In one embodiment, the method improves the Feed Conversion Ratio (FCR)of an animal by at least 1% and increases the proportion of bacteria ofgenus Faecalibacterium in the microbiota of the GI tract of an animal,wherein the bacteria of genus Faecalibacterium comprise 16S rRNA thathas at least 90% e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 13. In an embodiment,the proportion of bacteria of genus Faecalibacterium is increased by atleast 1%, such as at least 2%, at least 3%, at least 4% or at least 5%and the FCR is increased by at least 1.25%, preferably by at least 1.5%or most preferably by at least 1.75%.

In one embodiment, the method improves the Feed Conversion Ratio (FCR)of an animal by at least 1% and increases the proportion of bacteria ofgenus Faecalibacterium in the microbiota of the GI tract of an animal,wherein the bacteria of genus Faecalibacterium comprise 16S rRNA thathas at least 90% e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 13. In an embodiment,the proportion of bacteria of genus Faecalibacterium is increased byfactor of at least 1.25, such as at least 1.50, at least 1.75, at least2.0, at least 2.5 or at least 3.0 and the FCR is increased by at least1.25%, preferably by at least 1.5% or most preferably by at least 1.75%.

In a preferred embodiment, the invention relates to a method ofimproving the European Production Efficiency Factor (EPEF) and/or feedconversion ratio (FCR) of a monogastric animal comprising administeringan animal feed or animal feed additive comprising one or more microbiallysozymes to the monogastric animal, wherein:

(a) the microbial lysozyme is a microbial lysozyme comprising one ormore domains selected from the list consisting of GH24 and GH25, isdosed at a level of 10 to 150 ppm enzyme protein per kg animal feed andhas antimicrobial activity towards Clostridium perfringens;

(b) the monogastric animal is a selected from the group consisting ofswine, piglet, growing pig, sow, chicken, broiler, layer, pullet andchick;

(c) European Production Efficiency Factor (EPEF) and/or feed conversionratio (FCR) is improved by at least 1% compared to control; and

(d) optionally the microbial lysozyme is fed to the monogastric animalon a daily basis for at least 10 days during the life span of theanimal.

In one embodiment, the microbial lysozyme has at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 95% or at least100% of the antimicrobial activity of SEQ ID NO: 1 against Clostridiumperfringens under the conditions 50% MHB, pH 6. In another embodiment,the microbial lysozyme has at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 100% of theantimicrobial activity of SEQ ID NO: 4 against Clostridium perfringensunder the conditions 50% MHB, pH 6. In another embodiment, the microbiallysozyme has at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 100% of the antimicrobial activityof SEQ ID NO: 12 against Clostridium perfringens under the conditions50% MHB, pH 6.

In one embodiment, the EPEF is improved by at least 1.5%, such as by atleast 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or atleast 5% compared to the control. In another embodiment, the EPEF isimproved by between 1% and 15%, such as between 1% and 12%, between 1%and 10%, 1.5% and 8%, 2.0% and 7% compared to the control, or anycombination of these intervals.

In one embodiment, the FCR is improved by at least 1.25%, such as by atleast 1.5%, at least 1.75% or at least 2.0% compared to the control. Inanother embodiment, the FCR is improved by between 1% and 5%, such asbetween 1% and 4%, between 1% and 3%, 1.25% and 2.5%, 1.5% and 2%compared to the control, or any combination of these intervals.

In one embodiment, the microbial lysozyme is dosed at a level of 11 to125 ppm enzyme protein per kg animal feed, such as 12 to 100 ppm, 13 to75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to 60 ppmenzyme protein per kg animal feed, or any combination of theseintervals.

In one embodiment, the microbial lysozyme is fed to the animal frombirth until slaughter. In a preferred embodiment the microbial lysozymeis fed to the animal on a daily basis from birth until slaughter. Inanother preferred embodiment the microbial lysozyme is fed to the animalon a daily basis for at least 10 days, such as at least 15 days or atleast 20 days (where the days can be continuous or non-continuous)during the life span of the animal. In embodiment, the microbiallysozyme is fed to the animal for 10-20 days followed by a non-treatmentperiod of 5-10 days, and this cycle is repeated during the life span ofthe animal.

In a further embodiment, the microbial lysozyme is fed to broilers forthe first 49 days after hatching. In a further embodiment, the microbiallysozyme is fed to broilers for the first 36 days after hatching. In afurther embodiment, the microbial lysozyme is fed to broilers on days 22to 36 after hatching. In a further embodiment, the microbial lysozyme isfed to broilers during the pre-starter (days 1-7) period. In a furtherembodiment, the microbial lysozyme is fed to broilers during the starter(days 8-22) period. In a further embodiment, the microbial lysozyme isfed to broilers during the pre-starter (days 1-7) and starter (days8-22) period.

In a further embodiment, the microbial lysozyme is fed to layers duringthe life span of the animal. In a further embodiment, the microbiallysozyme is fed to layers for 76 weeks from hatching. In a furtherembodiment, the microbial lysozyme is fed to layers during the layingperiod, (from ca. week 18). In a further embodiment, the microbiallysozyme is fed to layers during the laying period but withheld duringthe forced molting period.

In a further embodiment, the microbial lysozyme is fed to turkeys duringlife span of the animal. In a further embodiment, the microbial lysozymeis fed to turkeys for 24 weeks from hatching. In a further embodiment,the microbial lysozyme is fed to turkeys for the first 16 weeks fromhatching (for hens) and for the first 20 weeks for hatching (for toms).

In a further embodiment, the microbial lysozyme is fed to swine duringlife span of the animal. In a further embodiment, the microbial lysozymeis fed to swine for 27 weeks from birth. In a further embodiment, themicrobial lysozyme is fed to piglets from birth to weaning (at 4 weeks).

In a further embodiment, the microbial lysozyme is fed to piglets forthe first 6 weeks from birth (4 weeks of lactation and 2 weekspost-weaning). In a further embodiment, the microbial lysozyme is fed toweaning piglets during the pre-starter (days 1-14 after weaning). In afurther embodiment, the microbial lysozyme is fed to weaning pigletsduring the starter (days 15-42 after weaning) period. In a furtherembodiment, the microbial lysozyme is fed to weaning piglets during thepre-starter (days 1-14 after weaning) and starter (days 15-42 afterweaning) period. In a further embodiment, the microbial lysozyme is fedto swine during the grower/fattening period (week 10 to ca. week 27after birth).

In one embodiment, the microbial lysozyme is of fungal origin. In anembodiment, the microbial lysozyme is obtained or obtainable from thephylum Ascomycota, such as the sub-phylum Pezizomycotina.

In one embodiment, the microbial lysozyme has at least 50%, e.g., atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 1.

In one embodiment, the microbial lysozyme comprises or consists of theamino acid sequence of SEQ ID NO: 1 or an allelic variant thereof; or isa fragment thereof having lysozyme activity, wherein the fragmentcomprises at least 170 amino acids, such as at least 175 amino acids, atleast 177 amino acids, at least 180 amino acids, at least 185 aminoacids, at least 190 amino acids, at least 195 amino acids or at least200 amino acids. In another embodiment, the microbial lysozyme comprisesor consists of the amino acid sequence of SEQ ID NO: 1 or an allelicvariant thereof and a N-terminal and/or C-terminal His-tag and/orHQ-tag. In another aspect, the polypeptide comprises or consists ofamino acids 1 to 213 of SEQ ID NO: 1.

In another embodiment, the microbial lysozyme is a variant of SEQ ID NO:1 wherein the variant has lysozyme activity and comprises one or moresubstitutions, and/or one or more deletions, and/or one or moreinsertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50 positions. In another embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 1 is between 1 and 45, such as1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 positions. In anembodiment, the number of positions comprising one or more amino acidsubstitutions, and/or one or more amino acid deletions, and/or one ormore amino acid insertions or any combination thereof in SEQ ID NO: 1 isnot more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In anotherembodiment, the number of substitutions, deletions, and/or insertions inSEQ ID NO: 1 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.In a further embodiment, the number of substitutions, preferablyconservative substitutions, in SEQ ID NO: 1 is not more than 10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a further embodiment, the number ofconservative substitutions in SEQ ID NO: 1 is not more than 10, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lysozyme activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

The crystal structure of the Acremonium alcalophilum CBS114.92 lysozymewas solved at a resolution of 1.3 Å as disclosed in WO 2013/076253.These atomic coordinates can be used to generate a three dimensionalmodel depicting the structure of the Acremonium alcalophilum CBS114.92lysozyme or homologous structures (such as the variants of the presentinvention). Using the x/ray structure, amino acid residues D95 and E97(using SEQ ID NO: 1 for numbering) were identified as catalyticresidues.

In one embodiment, the microbial lysozyme has at least 50%, e.g., atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 4.

In one embodiment, the microbial lysozyme comprises or consists of theamino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or isa fragment thereof having lysozyme activity, wherein the fragmentcomprises at least 210 amino acids, such as at least 215 amino acids, atleast 220 amino acids, at least 225 amino acids, at least 230 aminoacids, at least 235 amino acids or at least 240 amino acids. In anotherembodiment, the microbial lysozyme comprises or consists of the aminoacid sequence of SEQ ID NO: 4 or an allelic variant thereof and aN-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect,the polypeptide comprises or consists of amino acids 1 to 245 of SEQ IDNO: 4.

In another embodiment, the microbial lysozyme is a variant of SEQ ID NO:4 wherein the variant has lysozyme activity and comprises one or moresubstitutions, and/or one or more deletions, and/or one or moreinsertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50 positions. In another embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 4 is between 1 and 45, such as1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 positions. In anembodiment, the number of positions comprising one or more amino acidsubstitutions, and/or one or more amino acid deletions, and/or one ormore amino acid insertions or any combination thereof in SEQ ID NO: 4 isnot more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In anotherembodiment, the number of substitutions, deletions, and/or insertions inSEQ ID NO: 4 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.In a further embodiment, the number of substitutions, preferablyconservative substitutions, in SEQ ID NO: 4 is not more than 10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a further embodiment, the number ofconservative substitutions in SEQ ID NO: 4 is not more than 10, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.

Examples of amino acid changes, conservative substitutions and N- and/orC-terminal linkers are described above.

In one embodiment, the microbial lysozyme has at least 50%, e.g., atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to SEQ ID NO: 12.

In one embodiment, the microbial lysozyme comprises or consists of theamino acid sequence of SEQ ID NO: 12 or an allelic variant thereof; oris a fragment thereof having lysozyme activity, wherein the fragmentcomprises at least 210 amino acids, such as at least 215 amino acids, atleast 220 amino acids, at least 225 amino acids, at least 230 aminoacids, at least 235 amino acids or at least 240 amino acids. In anotherembodiment, the microbial lysozyme comprises or consists of the aminoacid sequence of SEQ ID NO: 12 or an allelic variant thereof and aN-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect,the polypeptide comprises or consists of amino acids 1 to 208 of SEQ IDNO: 12.

In another embodiment, the microbial lysozyme is a variant of SEQ ID NO:12 wherein the variant has lysozyme activity and comprises one or moresubstitutions, and/or one or more deletions, and/or one or moreinsertions or any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50 positions. In another embodiment, the number ofpositions comprising one or more amino acid substitutions, and/or one ormore amino acid deletions, and/or one or more amino acid insertions orany combination thereof in SEQ ID NO: 12 is between 1 and 45, such as1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5 positions. In anembodiment, the number of positions comprising one or more amino acidsubstitutions, and/or one or more amino acid deletions, and/or one ormore amino acid insertions or any combination thereof in SEQ ID NO: 12is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In anotherembodiment, the number of substitutions, deletions, and/or insertions inSEQ ID NO: 12 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or10. In a further embodiment, the number of substitutions, preferablyconservative substitutions, in SEQ ID NO: 12 is not more than 10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a further embodiment, the number ofconservative substitutions in SEQ ID NO: 12 is not more than 10, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Examples of amino acid changes, conservative substitutions and N- and/orC-terminal linkers are described above.

In another preferred embodiment, the invention relates to a method ofimproving the European Production Efficiency Factor (EPEF) and/or feedconversion ratio (FCR) of a monogastric animal comprising administeringan animal feed or animal feed additive comprising one or more microbiallysozymes to the monogastric animal, wherein:

(a) the microbial lysozyme is a GH24 lysozyme obtained or obtainablefrom the phylum Ascomycota, is dosed at a level of 10 to 150 ppm enzymeprotein per kg animal feed and has antimicrobial activity towardsClostridium perfringens;

(b) the monogastric animal is a selected from the group consisting ofswine, piglet, growing pig, sow, chicken, broiler, layer, pullet andchick;

(c) European Production Efficiency Factor (EPEF) and/or feed conversionratio (FCR) is improved by at least 1% compared to control; and

(d) the proportion of bacteria of genus Faecalibacterium in themicrobiota of the GI tract of an animal is increased by at least 1%.

In one embodiment, the EPEF is improved by at least 1.5%, such as by atleast 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or atleast 5% and the proportion of bacteria of genus Faecalibacterium isincreased by at least 2%, such as at least 3%, at least 4% or at least5% compared to the control.

In one embodiment, the FCR is improved by at least 1.25%, such as by atleast 1.5%, at least 1.75% or at least 2.0% and the proportion ofbacteria of genus Faecalibacterium is increased by at least 2%, such asat least 3%, at least 4% or at least 5% compared to the control.

In one embodiment, the microbial lysozyme is dosed at a level of 11 to125 ppm enzyme protein per kg animal feed, such as 12 to 100 ppm, 13 to75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to 60 ppmenzyme protein per kg animal feed, or any combination of theseintervals.

In one embodiment, the microbial lysozyme is fed to the animal using oneof the regimes as disclosed herein.

In another preferred embodiment, the invention relates to a method ofimproving the European Production Efficiency Factor (EPEF) and/or feedconversion ratio (FCR) of a monogastric animal comprising administeringan animal feed or animal feed additive comprising one or more microbiallysozymes to the monogastric animal, wherein:

(a) the microbial lysozyme is a GH24 lysozyme obtained or obtainablefrom the phylum Ascomycota, is dosed at a level of 10 to 150 ppm enzymeprotein per kg animal feed and has antimicrobial activity towardsClostridium perfringens;

(b) the monogastric animal is a selected from the group consisting ofswine, piglet, growing pig, sow, chicken, broiler, layer, pullet andchick;

(c) European Production Efficiency Factor (EPEF) and/or feed conversionratio (FCR) is improved by at least 1% compared to control; and

(d) the proportion of bacteria of genus Faecalibacterium in themicrobiota of the GI tract of an animal is increased by a factor of atleast 1.25.

In one embodiment, the EPEF is improved by at least 1.5%, such as by atleast 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or atleast 5% and the proportion of bacteria of genus Faecalibacterium isincreased by a factor of at least 1.50, such as at least 1.75, at least2.0, at least 2.5 or at least 3.0 compared to the control.

In one embodiment, the FCR is improved by at least 1.25%, such as by atleast 1.5%, at least 1.75% or at least 2.0% and the proportion ofbacteria of genus Faecalibacterium is increased by a factor of at least1.50, such as at least 1.75, at least 2.0, at least 2.5 or at least 3.0compared to the control.

In one embodiment, the microbial lysozyme is dosed at a level of 11 to125 ppm enzyme protein per kg animal feed, such as 12 to 100 ppm, 13 to75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to 60 ppmenzyme protein per kg animal feed, or any combination of theseintervals.

In one embodiment, the microbial lysozyme is fed to the animal using oneof the regimes as disclosed herein.

In another preferred embodiment, the invention relates to a method ofimproving the European Production Efficiency Factor (EPEF) and/or feedconversion ratio (FCR) of a monogastric animal comprising administeringan animal feed or animal feed additive comprising one or more microbiallysozymes to the monogastric animal, wherein:

(a) the microbial lysozyme is a GH25 lysozyme obtained or obtainablefrom the phylum Ascomycota, is dosed at a level of 10 to 150 ppm enzymeprotein per kg animal feed and has antimicrobial activity towardsClostridium perfringens;

(b) the monogastric animal is a selected from the group consisting ofswine, piglet, growing pig, sow, chicken, broiler, layer, pullet andchick;

(c) European Production Efficiency Factor (EPEF) and/or feed conversionratio (FCR) is improved by at least 1% compared to control; and

(d) the proportion of bacteria of genus Faecalibacterium in themicrobiota of the GI tract of an animal is increased by at least 1%.

In one embodiment, the EPEF is improved by at least 1.5%, such as by atleast 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or atleast 5% and the proportion of bacteria of genus Faecalibacterium isincreased by at least 2%, such as at least 3%, at least 4% or at least5% compared to the control.

In one embodiment, the FCR is improved by at least 1.25%, such as by atleast 1.5%, at least 1.75% or at least 2.0% and the proportion ofbacteria of genus Faecalibacterium is increased by at least 2%, such asat least 3%, at least 4% or at least 5% compared to the control.

In one embodiment, the microbial lysozyme is dosed at a level of 11 to125 ppm enzyme protein per kg animal feed, such as 12 to 100 ppm, 13 to75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to 60 ppmenzyme protein per kg animal feed, or any combination of theseintervals.

In one embodiment, the microbial lysozyme is fed to the animal using oneof the regimes as disclosed herein.

In another preferred embodiment, the invention relates to a method ofimproving the European Production Efficiency Factor (EPEF) and/or feedconversion ratio (FCR) of a monogastric animal comprising administeringan animal feed or animal feed additive comprising one or more microbiallysozymes to the monogastric animal, wherein:

(a) the microbial lysozyme is a GH25 lysozyme obtained or obtainablefrom the phylum Ascomycota, is dosed at a level of 10 to 150 ppm enzymeprotein per kg animal feed and has antimicrobial activity towardsClostridium perfringens;

(b) the monogastric animal is a selected from the group consisting ofswine, piglet, growing pig, sow, chicken, broiler, layer, pullet andchick;

-   -   (c) European Production Efficiency Factor (EPEF) and/or feed        conversion ratio (FCR) is improved by at least 1% compared to        control; and

(d) the proportion of bacteria of genus Faecalibacterium in themicrobiota of the GI tract of an animal is increased by a factor of atleast 1.25.

In one embodiment, the EPEF is improved by at least 1.5%, such as by atleast 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or atleast 5% and the proportion of bacteria of genus Faecalibacterium isincreased by a factor of at least 1.50, such as at least 1.75, at least2.0, at least 2.5 or at least 3.0 compared to the control.

In one embodiment, the FCR is improved by at least 1.25%, such as by atleast 1.5%, at least 1.75% or at least 2.0% and the proportion ofbacteria of genus Faecalibacterium is increased by a factor of at least1.50, such as at least 1.75, at least 2.0, at least 2.5 or at least 3.0compared to the control.

In one embodiment, the microbial lysozyme is dosed at a level of 11 to125 ppm enzyme protein per kg animal feed, such as 12 to 100 ppm, 13 to75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to 60 ppmenzyme protein per kg animal feed, or any combination of theseintervals.

In one embodiment, the microbial lysozyme is fed to the animal using oneof the regimes as disclosed herein.

Formulating Agent

The enzyme of the invention may be formulated as a liquid or a solid.For a liquid formulation, the formulating agent may comprise a polyol(such as e.g. glycerol, ethylene glycol or propylene glycol), a salt(such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or asugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, andsorbitol). Thus in one embodiment, the composition is a liquidcomposition comprising the polypeptide of the invention and one or moreformulating agents selected from the list consisting of glycerol,ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, sodiumchloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose,and sorbitol. The liquid formulation may be sprayed onto the feed afterit has been pelleted or may be added to drinking water given to theanimals.

For a solid formulation, the formulation may be for example as agranule, spray dried powder or agglomerate. The formulating agent maycomprise a salt (organic or inorganic zinc, sodium, potassium or calciumsalts such as e.g. such as calcium acetate, calcium benzoate, calciumcarbonate, calcium chloride, calcium citrate, calcium sorbate, calciumsulfate, potassium acetate, potassium benzoate, potassium carbonate,potassium chloride, potassium citrate, potassium sorbate, potassiumsulfate, sodium acetate, sodium benzoate, sodium carbonate, sodiumchloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate,zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zincsulfate), starch or a sugar or sugar derivative (such as e.g. sucrose,dextrin, glucose, lactose, sorbitol).

In an embodiment, the solid composition is in granulated form. Thegranule may have a matrix structure where the components are mixedhomogeneously. However, the granule typically comprises a core particleand one or more coatings, which typically are salt and/or wax coatings.Examples of waxes are polyethylene glycols; polypropylenes; Carnaubawax; Candelilla wax; bees wax; hydrogenated plant oil or animal tallowsuch as hydrogenated ox tallow, hydrogenated palm oil, hydrogenatedcotton seeds and/or hydrogenated soy bean oil; fatty acid alcohols;mono-glycerides and/or di-glycerides, such as glyceryl stearate, whereinstearate is a mixture of stearic and palmitic acid; micro-crystallinewax; paraffin's; and fatty acids, such as hydrogenated linear longchained fatty acids and derivatives thereof. A preferred wax is palm oilor hydrogenated palm oil. The core particle can either be a homogeneousblend of lysozyme of the invention optionally combined with one or moreadditional enzymes and optionally together with one or more salts or aninert particle with the lysozyme of the invention optionally combinedwith one or more additional enzymes applied onto it.

In an embodiment, the material of the core particles are selected fromthe group consisting of inorganic salts (such as calcium acetate,calcium benzoate, calcium carbonate, calcium chloride, calcium citrate,calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate,potassium carbonate, potassium chloride, potassium citrate, potassiumsorbate, potassium sulfate, sodium acetate, sodium benzoate, sodiumcarbonate, sodium chloride, sodium citrate, sodium sulfate, zincacetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate,zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (suchas e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugarderivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol),small organic molecules, starch, flour, cellulose and minerals and clayminerals (also known as hydrous aluminium phyllosilicates). In apreferred embodiment, the core comprises a clay mineral such askaolinite or kaolin.

The salt coating is typically at least 1 μm thick and can either be oneparticular salt or a mixture of salts, such as Na₂SO₄, K₂SO₄, MgSO₄and/or sodium citrate. Other examples are those described in e.g. WO2008/017659, WO 2006/034710, WO 1997/05245, WO 1998/54980, WO1998/55599, WO 2000/70034 or polymer coating such as described in WO2001/00042.

In another embodiment, the composition is a solid composition comprisingthe lysozyme of the invention and one or more formulating agentsselected from the list consisting of sodium chloride, sodium benzoate,potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate,sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose,sucrose, sorbitol, lactose, starch and cellulose. In a preferredembodiment, the formulating agent is selected from one or more of thefollowing compounds: sodium sulfate, dextrin, cellulose, sodiumthiosulfate and calcium carbonate. In a preferred embodiment, the solidcomposition is in granulated form. In an embodiment, the solidcomposition is in granulated form and comprises a core particle, anenzyme layer comprising the lysozyme of the invention and a saltcoating.

In a further embodiment, the formulating agent is selected from one ormore of the following compounds: glycerol, ethylene glycol, 1,2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodiumbenzoate, potassium sorbate, sodium sulfate, potassium sulfate,magnesium sulfate, sodium thiosulfate, calcium carbonate, sodiumcitrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolinand cellulose. In a preferred embodiment, the formulating agent isselected from one or more of the following compounds: 1, 2-propyleneglycol, 1, 3-propylene glycol, sodium sulfate, dextrin, cellulose,sodium thiosulfate, kaolin and calcium carbonate.

Animal Feed and Animal Feed Additives

Animal feed compositions or diets have a relatively high content ofprotein. Poultry and pig diets can be characterised as indicated inTable B of WO 2001/058275, columns 2-3. Fish diets can be characterisedas indicated in column 4 of this Table B. Furthermore such fish dietsusually have a crude fat content of 200-310 g/kg.

An animal feed composition according to the invention has a crudeprotein content of between 50 and 800 g/kg, and furthermore comprisesone or more polypeptides having lysozyme activity as described herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO2001/058275 (R. 2-5).

The nitrogen content is determined by the Kjeldahl method (A.O.A.C.,1984, Official Methods of Analysis 14th ed., Association of OfficialAnalytical Chemists, Washington D.C.) and crude protein is calculated asnitrogen (N) multiplied by a factor 6.25 (i.e. Crude protein (g/kg)=N(g/kg)×6.25).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen by, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein as defined above.

The animal feed composition of the invention may also contain animalprotein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal,typically in an amount of 0-25%. The animal feed composition of theinvention may also comprise Dried Distillers Grains with Solubles(DDGS), typically in amounts of 0-30%.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

The animal feed may comprise vegetable proteins. In particularembodiments, the protein content of the vegetable proteins is at least10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may bederived from vegetable protein sources, such as legumes and cereals, forexample, materials from plants of the families Fabaceae (Leguminosae),Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupinmeal, rapeseed meal, and combinations thereof.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean. In another particular embodiment, the vegetable proteinsource is material from one or more plants of the family Chenopodiaceae,e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetableprotein sources are rapeseed, and cabbage. In another particularembodiment, soybean is a preferred vegetable protein source. Otherexamples of vegetable protein sources are cereals such as barley, wheat,rye, oat, maize (corn), rice, and sorghum.

Animal diets can e.g. be manufactured as mash feed (non-pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, for mashfeed a solid or liquid enzyme formulation may be added before or duringthe ingredient mixing step. For pelleted feed the (liquid or solid)lysozyme/enzyme preparation may also be added before or during the feedingredient step. Typically a liquid enzyme preparation comprises thelysozyme of the invention optionally with a polyol, such as glycerol,ethylene glycol or propylene glycol, and is added after the pelletingstep, such as by spraying the liquid formulation onto the pellets. Thelysozyme may also be incorporated in a feed additive or premix.

Alternatively, the lysozyme can be prepared by freezing a mixture ofliquid enzyme solution with a bulking agent such as ground soybean meal,and then lyophilizing the mixture.

In an embodiment, the composition comprises one or more additionalenzymes. In an embodiment, the composition comprises one or moremicrobes. In an embodiment, the composition comprises one or morevitamins. In an embodiment, the composition comprises one or moreminerals. In an embodiment, the composition comprises one or more aminoacids. In an embodiment, the composition comprises one or more otherfeed ingredients.

In another embodiment, the composition comprises one or more of thepolypeptides of the invention, one or more formulating agents and one ormore additional enzymes. In an embodiment, the composition comprises oneor more of the polypeptides of the invention, one or more formulatingagents and one or more microbes. In an embodiment, the compositioncomprises one or more of the polypeptides of the invention, one or moreformulating agents and one or more vitamins. In an embodiment, thecomposition comprises one or more of the polypeptides of the inventionand one or more minerals. In an embodiment, the composition comprisesthe polypeptide of the invention, one or more formulating agents and oneor more amino acids. In an embodiment, the composition comprises one ormore of the polypeptides of the invention, one or more formulatingagents and one or more other feed ingredients.

In a further embodiment, the composition comprises one or more of thepolypeptides of the invention, one or more formulating agents and one ormore components selected from the list consisting of: one or moreadditional enzymes; one or more microbes; one or more vitamins; one ormore minerals; one or more amino acids; and one or more other feedingredients.

The final lysozyme concentration in the diet is within the range of0.01-200 ppm enzyme protein per kg animal feed, such as 0.1 to 150 ppm,0.5 to 100 ppm, 1 to 75 ppm, 2 to 50 ppm, 3 to 25 ppm, 2 to 80 ppm, 5 to60 ppm, 8 to 40 ppm or 10 to 30 ppm enzyme protein per kg animal feed,or any combination of these intervals.

It is at present contemplated that the lysozyme is administered in oneor more of the following amounts (dosage ranges): 0.01-200; 0.01-100;0.5-100; 1-50; 5-100; 5-50; 10-100; 0.05-50; 5-25; or 0.10-10—all theseranges being in mg lysozyme per kg feed (ppm).

For determining mg lysozyme protein per kg feed, the lysozyme ispurified from the feed composition, and the specific activity of thepurified lysozyme is determined using a relevant assay (see underlysozyme activity). The lysozyme activity of the feed composition assuch is also determined using the same assay, and on the basis of thesetwo determinations, the dosage in mg lysozyme protein per kg feed iscalculated.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The same principles apply for determining mg lysozyme protein in feedadditives. Of course, if a sample is available of the lysozyme used forpreparing the feed additive or the feed, the specific activity isdetermined from this sample (no need to purify the lysozyme from thefeed composition or the additive).

Additional Enzymes

In another embodiment, the compositions described herein optionallyinclude one or more enzymes. Enzymes can be classified on the basis ofthe handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also theENZYME site at the internet: http://www.expasy.ch/enzyme/. ENZYME is arepository of information relative to the nomenclature of enzymes. It isprimarily based on the recommendations of the Nomenclature Committee ofthe International Union of Biochemistry and Molecular Biology (IUB-MB),Academic Press, Inc., 1992, and it describes each type of characterizedenzyme for which an EC (Enzyme Commission) number has been provided(Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305).This IUB-MB Enzyme nomenclature is based on their substrate specificityand occasionally on their molecular mechanism; such a classificationdoes not reflect the structural features of these enzymes.

Another classification of certain glycoside hydrolase enzymes, such asendoglucanase, xylanase, galactanase, mannanase, dextranase, lysozymeand galactosidase is described in Henrissat et al, “Thecarbohydrate-active enzymes database (CAZy) in 2013”, Nucl. Acids Res.(1 Jan. 2014) 42 (D1): D490-D495; see also www.cazy.org.

Thus the composition of the invention may also comprise at least oneother enzyme selected from the group comprising of xylanase (EC3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22);protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3);phospholipase D (EC 3.1.4.4); amylase such as, for example,alpha-amylase (EC 3.2.1.1); arabinofuranosidase (EC 3.2.1.55);beta-xylosidase (EC 3.2.1.37); acetyl xylan esterase (EC 3.1.1.72);feruloyl esterase (EC 3.1.1.73); cellulase (EC 3.2.1.4);cellobiohydrolases (EC 3.2.1.91); beta-glucosidase (EC 3.2.1.21);pullulanase (EC 3.2.1.41), alpha-mannosidase (EC 3.2.1.24), mannanase(EC 3.2.1.25) and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or anycombination thereof.

In a particular embodiment, the composition of the invention comprises aphytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially availablephytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P, Ronozyme®NP and Ronozyme® HiPhos (DSM Nutritional Products), Natuphos™ (BASF),Finase® and Quantum® Blue (AB Enzymes), OptiPhos® (Huvepharma) Phyzyme®XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytasesinclude those described in e.g. WO 98/28408, WO 00/43503, and WO03/066847.

In a particular embodiment, the composition of the invention comprises axylanase (EC 3.2.1.8). Examples of commercially available xylanasesinclude Ronozyme® WX and Ronozyme® G2 (DSM Nutritional Products),Econase® XT and Barley (AB Vista), Xylathin® (Verenium), Hostazym® X(Huvepharma) and Axtra® XB (Xylanase/beta-glucanase, DuPont).

In a particular embodiment, the composition of the invention comprises aprotease (EC 3.4). Examples of commercially available proteases includeRonozyme® ProAct (DSM Nutritional Products).

Microbes

In an embodiment, the animal feed composition further comprises one ormore additional microbes. In a particular embodiment, the animal feedcomposition further comprises a bacterium from one or more of thefollowing genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus,Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or anycombination thereof.

In a preferred embodiment, animal feed composition further comprises abacterium from one or more of the following strains: Bacillus subtilis,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus,Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, Enterococcus faecium, Enterococcus spp,and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp,Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis,Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillusfarciminus, lactobacillus rhamnosus, Clostridium butyricum,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri,Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii,Propionibacteria sp.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus subtilis: 3A-P4 (PTA-6506), 15A-P4(PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01(NRRL-B-50104), BS27 (NRRL B-501 05), BS 18 (NRRL B-50633), BS 278 (NRRLB-50634), DSM 29870, DSM 29871, NRRL B-50136, NRRL B-50605, NRRLB-50606, NRRL B-50622 and PTA-7547.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus pumilus: NRRL B-50016, ATCC 700385, NRRLB-50885 or NRRL B-50886.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus lichenformis: NRRL B 50015, NRRL B-50621or NRRL B-50623.

In a more preferred embodiment, composition, animal feed additive oranimal feed further comprises a bacterium from one or more of thefollowing strains of Bacillus amyloliquefaciens: DSM 29869, DSM 29872,NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B-50606, NRRLB-50013, NRRL B-50151, NRRL B-50141, NRRL B-50147 or NRRL B-50888.

The bacterial count of each of the bacterial strains in the animal feedcomposition is between 1×10⁴ and 1×10¹⁴ CFU/kg of dry matter, preferablybetween 1×10⁶ and 1×10¹² CFU/kg of dry matter, and more preferablybetween 1×10⁷ and 1×10¹¹ CFU/kg of dry matter. In a more preferredembodiment the bacterial count of each of the bacterial strains in theanimal feed composition is between 1×10⁸ and 1×10¹⁰ CFU/kg of drymatter.

The bacterial count of each of the bacterial strains in the animal feedcomposition is between 1×10⁵ and 1×10¹⁵ CFU/animal/day, preferablybetween 1×10⁷ and 1×10¹³ CFU/animal/day, and more preferably between1×10⁸ and 1×10¹² CFU/animal/day. In a more preferred embodiment thebacterial count of each of the bacterial strains in the animal feedcomposition is between 1×10⁹ and 1×10¹¹ CFU/animal/day.

In another embodiment, the one or more bacterial strains are present inthe form of a stable spore.

Premix

In an embodiment, the animal feed may include a premix, comprising e.g.vitamins, minerals, enzymes, amino acids, preservatives, antibiotics,other feed ingredients or any combination thereof which are mixed intothe animal feed.

Amino Acids

The composition of the invention may further comprise one or more aminoacids. Examples of amino acids which are used in animal feed are lysine,alanine, beta-alanine, threonine, methionine and tryptophan.

Vitamins and Minerals

In another embodiment, the animal feed may include one or more vitamins,such as one or more fat-soluble vitamins and/or one or morewater-soluble vitamins. In another embodiment, the animal feed mayoptionally include one or more minerals, such as one or more traceminerals and/or one or more macro minerals.

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed.

Non-limiting examples of fat-soluble vitamins include vitamin A, vitaminD3, vitamin E, and vitamin K, e.g., vitamin K3.

Non-limiting examples of water-soluble vitamins include vitamin B12,biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folicacid and panthothenate, e.g., Ca-D-panthothenate.

Non-limiting examples of trace minerals include boron, cobalt, chloride,chromium, copper, fluoride, iodine, iron, manganese, molybdenum,selenium and zinc.

Non-limiting examples of macro minerals include calcium, magnesium,potassium and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 2001/058275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

In a still further embodiment, the animal feed additive of the inventioncomprises at least one of the below vitamins, preferably to provide anin-feed-concentration within the ranges specified in the below Table 1(for piglet and broiler diets, respectively).

TABLE 1 Typical vitamin recommendations Vitamin Piglet diet Broiler dietVitamin A 10,000-15,000 IU/kg feed 8-12,500 IU/kg feed Vitamin D31800-2000 IU/kg feed 3000-5000 IU/kg feed Vitamin E 60-100 mg/kg feed150-240 mg/kg feed Vitamin K3 2-4 mg/kg feed 2-4 mg/kg feed Vitamin B12-4 mg/kg feed 2-3 mg/kg feed Vitamin B2 6-10 mg/kg feed 7-9 mg/kg feedVitamin B6 4-8 mg/kg feed 3-6 mg/kg feed Vitamin B12 0.03-0.05 mg/kgfeed 0.015-0.04 mg/kg feed Niacin (Vitamin B3) 30-50 mg/kg feed 50-80mg/kg feed Pantothenic acid 20-40 mg/kg feed 10-18 mg/kg feed Folic acid1-2 mg/kg feed 1-2 mg/kg feed Biotin 0.15-0.4 mg/kg feed 0.15-0.3 mg/kgfeed Choline chloride 200-400 mg/kg feed 300-600 mg/kg feed

Other Feed Ingredients

The composition of the invention may further comprise colouring agents,stabilisers, growth improving additives and aroma compounds/flavourings,polyunsaturated fatty acids (PUFAs);

reactive oxygen generating species, anti-microbial peptides andanti-fungal polypeptides. Examples of colouring agents are carotenoidssuch as beta-carotene, astaxanthin, and lutein.

Examples of aroma compounds/flavourings are creosol, anethol, deca-,undeca- and/or dodeca-lactones, ionones, irone, gingerol, piperidine,propylidene phatalide, butylidene phatalide, capsaicin and tannin.

Examples of stabilizing agents (e.g. acidifiers) are organic acids.Examples of these are benzoic acid (VevoVitall®, DSM NutritionalProducts), formic acid, butyric acid, fumaric acid and propionic acid.

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and enzymes such as an oxidase,an oxygenase or a syntethase.

The composition of the invention may further comprise at least one aminoacid. Examples of amino acids which are used in animal feed are lysine,alanine, beta-alanine, threonine, methionine and tryptophan.

Use of Microbial Lyzozyme to Improve Animal Performance

In another aspect, the invention relates to the use of an animal feedadditive or an animal feed for improving the European ProductionEfficiency Factor (EPEF) and/or Feed Conversion Ratio (FCR) in amonogastric animal wherein the animal feed or animal feed additivecomprises one or more microbial lysozymes, wherein the microbiallysozyme is administered at a level of 8 to 250 ppm enzyme protein perkg animal feed.

In a preferred embodiment, the improvement is compared to an animal feedor animal feed additive wherein the microbial lysozyme is not present(herein referred to as the negative control).

In one embodiment, the EPEF is improved by at least 1%, such as by atleast 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least 3.5%, atleast 4% or at least 5% compared to the control.

In another embodiment, the EPEF is improved by between 1% and 15%, suchas between 1% and 12%, between 1% and 10%, 1.5% and 8%, 2.0% and 7%compared to the control, or any combination of these intervals.

In one embodiment, the FCR is improved by at least 1%, such as by atleast 1.25%, at least 1.5%, at least 1.75% or at least 2.0% compared tothe control. In another embodiment, the FCR is improved by between 1%and 5%, such as between 1% and 4%, between 1% and 3%, 1.25% and 2.5%,1.5% and 2% compared to the control, or any combination of theseintervals.

In one embodiment, the microbial lysozyme is dosed at a level of 9 to200 ppm enzyme protein per kg animal feed, such as 10 to 150 ppm, 11 to125 ppm, 12 to 100 ppm, 13 to 75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25to 75 ppm or 30 to 60 ppm enzyme protein per kg animal feed, or anycombination of these intervals.

In one embodiment, the monogastric animal is selected from the groupconsisting of swine, piglet, growing pig, sow, poultry, turkey, duck,quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer,pullet and chick, horse, crustaceans, shrimps, prawns, fish, amberjack,arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama,carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie,dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut,java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet,paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa,sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook,sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia,trout, tuna, turbot, vendace, walleye and whitefish. In a preferredembodiment, the monogastric animal is selected from the group consistingof swine, piglet, growing pig, sow, poultry, turkey, duck, quail, guineafowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick.In a more preferred embodiment, the monogastric animal is selected fromthe group consisting of swine, piglet, growing pig, sow, chicken,broiler, layer, pullet and chick.

In one embodiment, the microbial lysozyme has antimicrobial activitytowards Clostridium perfringens. In an embodiment, the microbiallysozyme has at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 100% of the antimicrobial activityof SEQ ID NO: 1 against Clostridium perfringens under the conditions 50%MHB, pH 6. In an embodiment, the microbial lysozyme has at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 100% of the antimicrobial activity of SEQ ID NO: 4 againstClostridium perfringens under the conditions 50% MHB, pH 6. In anembodiment, the microbial lysozyme has at least 60%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95% or at least 100% ofthe antimicrobial activity of SEQ ID NO: 12 against Clostridiumperfringens under the conditions 50% MHB, pH 6. Antimicrobial activitytowards Clostridium perfringens can be determined according to theantimicrobial assay described in Example 6.

In another embodiment, the invention relates to a composition comprisinga microbial lysozyme for the treatment of Clostridium perfringens in amonogastric animal wherein the composition improves the EuropeanProduction Efficiency Factor (EPEF) and/or feed conversion ratio (FCR)of the monogastric animal.

In one embodiment, the microbial lysozyme is fed to the animal frombirth until slaughter. In a preferred embodiment the microbial lysozymeis fed to the animal on a daily basis from birth until slaughter. Inanother preferred embodiment the microbial lysozyme is fed to the animalon a daily basis for at least 10 days, such as at least 15 days or atleast 20 days (where the days can be continuous or non-continuous)during the life span of the animal. In another embodiment, the microbiallysozyme is fed to the animal for 10-20 days followed by a non-treatmentperiod of 5-10 days, and this cycle is repeated during the life span ofthe animal.

In a further embodiment, the microbial lysozyme is fed to broilers forthe first 49 days after hatching. In a further embodiment, the microbiallysozyme is fed to broilers for the first 36 days after hatching. In afurther embodiment, the microbial lysozyme is fed to broilers on days 22to 36 after hatching. In a further embodiment, the microbial lysozyme isfed to broilers during the pre-starter (days 1-7) period. In a furtherembodiment, the microbial lysozyme is fed to broilers during the starter(days 8-22) period. In a further embodiment, the microbial lysozyme isfed to broilers during the pre-starter (days 1-7) and starter (days8-22) period.

In a further embodiment, the microbial lysozyme is fed to layers duringthe life span of the animal. In a further embodiment, the microbiallysozyme is fed to layers for 76 weeks from hatching. In a furtherembodiment, the microbial lysozyme is fed to layers during the layingperiod, (from ca. week 18). In a further embodiment, the microbiallysozyme is fed to layers during the laying period but withheld duringthe forced molting period.

In a further embodiment, the microbial lysozyme is fed to turkeys duringlife span of the animal. In a further embodiment, the microbial lysozymeis fed to turkeys for 24 weeks from hatching. In a further embodiment,the microbial lysozyme is fed to turkeys for the first 16 weeks fromhatching (for hens) and for the first 20 weeks for hatching (for toms).

In a further embodiment, the microbial lysozyme is fed to swine duringlife span of the animal. In a further embodiment, the microbial lysozymeis fed to swine for 27 weeks from birth. In a further embodiment, themicrobial lysozyme is fed to piglets from birth to weaning (at 4 weeks).In a further embodiment, the microbial lysozyme is fed to piglets forthe first 6 weeks from birth (4 weeks of lactation and 2 weekspost-weaning). In a further embodiment, the microbial lysozyme is fed toweaning piglets during the pre-starter (days 1-14 after weaning). In afurther embodiment, the microbial lysozyme is fed to weaning pigletsduring the starter (days 15-42 after weaning) period. In a furtherembodiment, the microbial lysozyme is fed to weaning piglets during thepre-starter (days 1-14 after weaning) and starter (days 15-42 afterweaning) period. In a further embodiment, the microbial lysozyme is fedto swine during the grower/fattening period (week 10 to ca. week 27after birth).

In one embodiment, the microbial lysozyme is of fungal origin. In anembodiment, the microbial lysozyme is obtained or obtainable from thephylum Ascomycota, such as the sub-phylum Pezizomycotina.

In one embodiment, the microbial lysozyme comprises one or more domainsselected from the list consisting of GH24 and GH25.

Methods of Altering the Population of Bacteria in the GI Tract of anAnimal

In one aspect, the invention relates to a method of increasing theproportion of bacteria of genus Faecalibacterium in the microbiome ofthe GI tract of a monogastric animal comprising administering to theanimal an animal feed or animal feed additive comprising one or moremicrobial lysozymes administered at a level of 8 to 250 ppm enzymeprotein per kg animal feed.

In one embodiment, the proportion of bacteria of genus Faecalibacteriumis increased by factor of at least 1.25, such as at least 1.50, at least1.75, at least 2.0, at least 2.5 or at least 3.0.

In one embodiment, the proportion of bacteria of genus Faecalibacteriumis increased by at least 1%, such as at least 2%, at least 3%, at least4% or at least 5%.

In one embodiment, the microbial lysozyme is dosed at a level of 11 to125 ppm enzyme protein per kg animal feed, such as 12 to 100 ppm, 13 to75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to 60 ppmenzyme protein per kg animal feed, or any combination of theseintervals.

In one embodiment, the microbial lysozyme is fed to the animal using oneof the regimes as disclosed herein.

In one embodiment, the microbial lysozyme is of fungal origin. In anembodiment, the microbial lysozyme is obtained or obtainable from thephylum Ascomycota, such as the sub-phylum Pezizomycotina.

In one embodiment, the microbial lysozyme comprises one or more domainsselected from the list consisting of GH24 and GH25.

In one embodiment, the method increases the proportion of bacteria ofgenus Faecalibacterium in the microbiota of the GI tract of an animal,wherein the bacteria of genus Faecalibacterium comprise 16S rRNA thathas at least 90% e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 13. In an embodiment,the proportion of bacteria of genus Faecalibacterium is increased by atleast 1%, such as at least 2%, at least 3%, at least 4% or at least 5%.In an alternative embodiment, the proportion of bacteria of genusFaecalibacterium is increased by a factor of at least 1.25, such as atleast 1.50, at least 1.75, at least 2.0, at least 2.5 or at least 3.0.

In one embodiment, the method improves the European ProductionEfficiency Factor (EPEF) of an animal by at least 1% and increases theproportion of bacteria of genus Faecalibacterium in the microbiota ofthe GI tract of an animal, wherein the bacteria of genusFaecalibacterium comprise 16S rRNA that has at least 90% e.g., at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto SEQ ID NO: 13. In an embodiment, the proportion of bacteria of genusFaecalibacterium is increased by at least 1%, such as at least 2%, atleast 3%, at least 4% or at least 5% and the EPEF is increased by atleast 1.25%, preferably by at least 1.5%, at least 1.75%, at least 2%,at least 3%, at least 4% or most preferably by at least 5%.

In one embodiment, the method improves the European ProductionEfficiency Factor (EPEF) of an animal by at least 1% and increases theproportion of bacteria of genus Faecalibacterium in the microbiota ofthe GI tract of an animal, wherein the bacteria of genusFaecalibacterium comprise 16S rRNA that has at least 90% e.g., at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identityto SEQ ID NO: 13. In an embodiment, the proportion of bacteria of genusFaecalibacterium is increased by factor of at least 1.25, such as atleast 1.50, at least 1.75, at least 2.0, at least 2.5 or at least 3.0and the EPEF is increased by at least 1.25%, preferably by at least1.5%, at least 1.75%, at least 2%, at least 3%, at least 4% or mostpreferably by at least 5%.

In one embodiment, the method improves the Feed Conversion Ratio (FCR)of an animal by at least 1% and increases the proportion of bacteria ofgenus Faecalibacterium in the microbiota of the GI tract of an animal,wherein the bacteria of genus Faecalibacterium comprise 16S rRNA thathas at least 90% e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 13. In an embodiment,the proportion of bacteria of genus Faecalibacterium is increased by atleast 1%, such as at least 2%, at least 3%, at least 4% or at least 5%and the FCR is increased by at least 1.25%, preferably by at least 1.5%or most preferably by at least 1.75%.

In one embodiment, the method improves the Feed Conversion Ratio (FCR)of an animal by at least 1% and increases the proportion of bacteria ofgenus Faecalibacterium in the microbiota of the GI tract of an animal,wherein the bacteria of genus Faecalibacterium comprise 16S rRNA thathas at least 90% e.g., at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 13. In an embodiment,the proportion of bacteria of genus Faecalibacterium is increased byfactor of at least 1.25, such as at least 1.50, at least 1.75, at least2.0, at least 2.5 or at least 3.0 and the FCR is increased by at least1.25%, preferably by at least 1.5% or most preferably by at least 1.75%.

In one preferred embodiment, the invention relates to a method ofincreasing the proportion of bacteria of genus Faecalibacterium in themicrobiome of the GI tract of a monogastric animal comprisingadministering to the animal an animal feed or animal feed additivecomprising one or more microbial lysozymes administered at a level of 10to 150 ppm enzyme protein per kg animal feed, wherein:

(a) the microbial lysozyme is a GH24 lysozyme obtained or obtainablefrom the phylum Ascomycota, preferably the sub-phylum Pezizomycotina;

(b) the monogastric animal is a selected from the group consisting ofswine, piglet, growing pig, sow, chicken, broiler, layer, pullet andchick; and

(c) the proportion of bacteria of genus Faecalibacterium in themicrobiota of the GI tract of an animal is increased by at least 1%.

In one embodiment, the proportion of bacteria of genus Faecalibacteriumis increased by at least 2%, such as at least 3%, at least 4% or atleast 5%. In one embodiment, the microbial lysozyme is dosed at a levelof 11 to 125 ppm enzyme protein per kg animal feed, such as 12 to 100ppm, 13 to 75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to60 ppm enzyme protein per kg animal feed, or any combination of theseintervals. In one embodiment, the microbial lysozyme is fed to theanimal using one of the regimes as disclosed herein.

In one embodiment, the method further improves EPEF by at least 1%, suchas by at least 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least3.5%, at least 4% or at least 5% compared to the control. In anotherembodiment, the method further improves EPEF by between 1% and 15%, suchas between 1% and 12%, between 1% and 10%, 1.5% and 8%, 2.0% and 7%compared to the control, or any combination of these intervals.

In one embodiment, the method further improves FCR by at least 1%, suchas by at least 1.25%, at least 1.5%, at least 1.75% or at least 2.0%compared to the control. In another embodiment, the method furtherimproves FCR by between 1% and 5%, such as between 1% and 4%, between 1%and 3%, 1.25% and 2.5%, 1.5% and 2% compared to the control, or anycombination of these intervals.

In one preferred embodiment, the invention relates to a method ofincreasing the proportion of bacteria of genus Faecalibacterium in themicrobiome of the GI tract of a monogastric animal comprisingadministering to the animal an animal feed or animal feed additivecomprising one or more microbial lysozymes administered at a level of 10to 150 ppm enzyme protein per kg animal feed, wherein:

(a) the microbial lysozyme is a GH25 lysozyme obtained or obtainablefrom the phylum Ascomycota, preferably the sub-phylum Pezizomycotina;

(b) the monogastric animal is a selected from the group consisting ofswine, piglet, growing pig, sow, chicken, broiler, layer, pullet andchick; and

(c) the proportion of bacteria of genus Faecalibacterium in themicrobiota of the GI tract of an animal is increased by at least 1%.

In one embodiment, the proportion of bacteria of genus Faecalibacteriumis increased by at least 2%, such as at least 3%, at least 4% or atleast 5%. In one embodiment, the microbial lysozyme is dosed at a levelof 11 to 125 ppm enzyme protein per kg animal feed, such as 12 to 100ppm, 13 to 75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to60 ppm enzyme protein per kg animal feed, or any combination of theseintervals. In one embodiment, the microbial lysozyme is fed to theanimal using one of the regimes as disclosed herein.

In one embodiment, the method further improves EPEF by at least 1%, suchas by at least 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least3.5%, at least 4% or at least 5% compared to the control. In anotherembodiment, the method further improves EPEF by between 1% and 15%, suchas between 1% and 12%, between 1% and 10%, 1.5% and 8%, 2.0% and 7%compared to the control, or any combination of these intervals.

In one embodiment, the method further improves FCR by at least 1%, suchas by at least 1.25%, at least 1.5%, at least 1.75% or at least 2.0%compared to the control. In another embodiment, the method furtherimproves FCR by between 1% and 5%, such as between 1% and 4%, between 1%and 3%, 1.25% and 2.5%, 1.5% and 2% compared to the control, or anycombination of these intervals.

In one preferred embodiment, the invention relates to a method ofincreasing the proportion of bacteria of genus Faecalibacterium in themicrobiome of the GI tract of a monogastric animal comprisingadministering to the animal an animal feed or animal feed additivecomprising one or more microbial lysozymes administered at a level of 10to 150 ppm enzyme protein per kg animal feed, wherein:

(a) the microbial lysozyme is a GH24 lysozyme obtained or obtainablefrom the phylum Ascomycota, preferably the sub-phylum Pezizomycotina;

(b) the monogastric animal is a selected from the group consisting ofswine, piglet, growing pig, sow, chicken, broiler, layer, pullet andchick;

(c) the proportion of bacteria of genus Faecalibacterium in themicrobiota of the GI tract of an animal is increased by at least 1%; and

(d) the bacteria of genus Faecalibacterium comprise 16S rRNA that has atleast 90% e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 13.

In one embodiment, the proportion of bacteria of genus Faecalibacteriumis increased by at least 2%, such as at least 3%, at least 4% or atleast 5%. In one embodiment, the microbial lysozyme is dosed at a levelof 11 to 125 ppm enzyme protein per kg animal feed, such as 12 to 100ppm, 13 to 75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to60 ppm enzyme protein per kg animal feed, or any combination of theseintervals. In one embodiment, the microbial lysozyme is fed to theanimal using one of the regimes as disclosed herein.

In one embodiment, the method further improves EPEF by at least 1%, suchas by at least 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least3.5%, at least 4% or at least 5% compared to the control. In anotherembodiment, the method further improves EPEF by between 1% and 15%, suchas between 1% and 12%, between 1% and 10%, 1.5% and 8%, 2.0% and 7%compared to the control, or any combination of these intervals.

In one embodiment, the method further improves FCR by at least 1%, suchas by at least 1.25%, at least 1.5%, at least 1.75% or at least 2.0%compared to the control. In another embodiment, the method furtherimproves FCR by between 1% and 5%, such as between 1% and 4%, between 1%and 3%, 1.25% and 2.5%, 1.5% and 2% compared to the control, or anycombination of these intervals.

In one preferred embodiment, the invention relates to a method ofincreasing the proportion of bacteria of genus Faecalibacterium in themicrobiome of the GI tract of a monogastric animal comprisingadministering to the animal an animal feed or animal feed additivecomprising one or more microbial lysozymes administered at a level of 10to 150 ppm enzyme protein per kg animal feed, wherein:

(a) the microbial lysozyme is a GH25 lysozyme obtained or obtainablefrom the phylum Ascomycota, preferably the sub-phylum Pezizomycotina;

(b) the monogastric animal is a selected from the group consisting ofswine, piglet, growing pig, sow, chicken, broiler, layer, pullet andchick;

(c) the proportion of bacteria of genus Faecalibacterium in themicrobiota of the GI tract of an animal is increased by at least 1%; and

(d) the bacteria of genus Faecalibacterium comprise 16S rRNA that has atleast 90% e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 13.

In one embodiment, the proportion of bacteria of genus Faecalibacteriumis increased by at least 2%, such as at least 3%, at least 4% or atleast 5%. In one embodiment, the microbial lysozyme is dosed at a levelof 11 to 125 ppm enzyme protein per kg animal feed, such as 12 to 100ppm, 13 to 75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to60 ppm enzyme protein per kg animal feed, or any combination of theseintervals. In one embodiment, the microbial lysozyme is fed to theanimal using one of the regimes as disclosed herein.

In one embodiment, the method further improves EPEF by at least 1%, suchas by at least 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least3.5%, at least 4% or at least 5% compared to the control. In anotherembodiment, the method further improves EPEF by between 1% and 15%, suchas between 1% and 12%, between 1% and 10%, 1.5% and 8%, 2.0% and 7%compared to the control, or any combination of these intervals.

In one embodiment, the method further improves FCR by at least 1%, suchas by at least 1.25%, at least 1.5%, at least 1.75% or at least 2.0%compared to the control. In another embodiment, the method furtherimproves FCR by between 1% and 5%, such as between 1% and 4%, between 1%and 3%, 1.25% and 2.5%, 1.5% and 2% compared to the control, or anycombination of these intervals.

EXAMPLES Strains

Trichophaea saccata CBS804.70 was purchased from the Centraalbureau voorSchimmelcultures (Utrecht, the Netherlands). According to Central Bureauvor Schnimmelkulture, Trichophaea saccata CBS804.70 was isolated fromcoal spoil tip soil from Staffordshire, England in May 1968.

According to Central Bureau vor Schnimmelkulture, Acremoniumalcalophilum CBS 114.92 was isolated by A. Yoneda in 1984 from thesludge of pig faeces compost near Tsukui Lake, Japan.

Media and Solutions

YP+2% glucose medium was composed of 1% yeast extract, 2% peptone and 2%glucose.

YP+2% maltodextrin medium was composed of 1% yeast extract, 2% peptoneand 2% maltodextrin.

PDA agar plates were composed of potato infusion (potato infusion wasmade by boiling 300 g of sliced (washed but unpeeled) potatoes in waterfor 30 minutes and then decanting or straining the broth throughcheesecloth. Distilled water was then added until the total volume ofthe suspension was one liter, followed by 20 g of dextrose and 20 g ofagar powder. The medium was sterilized by autoclaving at 15 psi for 15minutes (Bacteriological Analytical Manual, 8th Edition, Revision A,1998).

LB plates were composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1liter.

LB medium was composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, and deionized water to 1 liter.

COVE sucrose plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 ml of COVE salts solution, and deionized water to 1 liter.The medium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and 10 mM acetamide, 15 mM CsCl, TRITON®X-100 (50 μl/500 ml) were added.

COVE salts solution was composed of 26 g of MgSO4.7H2O, 26 g of KCL, 26g of KH2PO4, 50 ml of COVE trace metals solution, and deionized water to1 liter.

COVE trace metals solution was composed of 0.04 g of Na2B4O7.10H2O, 0.4g of CuSO4.5H2O, 1.2 g of FeSO4.7H2O, 0.7 g of MnSO4.H2O, 0.8 g ofNa2MoO4.2H2O, 10 g of ZnSO4.7H2O, and deionized water to 1 liter.

Example 1: Cloning, Expression and Purification of the GH25 Lysozymefrom Acremonium alcalophilum CBS 114.92

The GH25 lysozyme from Acremonium alcalophilum CBS 114.92 (SEQ ID NO: 1)was cloned and expressed as described in example 8 and purified asdescribed in example 5 of WO 2013/076253. Alternatively, SEQ ID NO: 12can be cloned and expressed as described in example 2 of WO 2013/076253.

Example 2: Expression of the GH24 Lysozyme from Trichophaea saccata

The fungal strain was cultivated in 100 ml of YP+2% glucose medium in1000 ml Erlenmeyer shake flasks for 5 days at 20° C. Mycelia wereharvested from the flasks by filtration of the medium through a Buchnervacuum funnel lined with MIRACLOTH® (EMD Millipore, Billerica, Mass.,USA). Mycelia were frozen in liquid nitrogen and stored at −80° C. untilfurther use. Genomic DNA was isolated using a DNEASY® Plant Maxi Kit(QIAGEN GMBH, Hilden Germany) according to the manufacturer'sinstructions.

Genomic sequence information was generated by Illumina MySeq (IlluminaInc., San Diego, Calif.). 5 μgs of the isolated Trichophaea saccatagenomic DNA was used for library preparation and analysis according tothe manufacturer's instructions. A 100 bp, paired end strategy wasemployed with a library insert size of 200-500 bp. One half of a HiSeqrun was used for the total of 95,744,298, 100 bp raw reads obtained. Thereads were subsequently fractionated to 25% followed by trimming(extracting longest sub-sequences having Phred-scores of 10 or more).These reads were assembled using Idba version 0.19. Contigs shorter than400 bp were discarded, resulting in 8,954,791,030 bp with an N-50 of10,035. Genes were called using GeneMark.hmm ES version 2.3c andidentification of the catalytic domain was made using “Phage lysozymePF00959” Hidden Markov Model provided by Pfam. The polypeptide codingsequence for the entire coding region was cloned from Trichophaeasaccata CBS804.70 genomic DNA by PCR using the primers F-80470 andR-80470 (SEQ ID NO: 6 and SEQ ID NO: 7 respectively) as described below.

(SEQ ID NO: 6) 5′-ACACAACTGGGGATCCACCATGCACGCTCTCACCCTTCT-3′(SEQ ID NO: 7) 5′-CTAGATCTCGAGAAGCTT TTAGCACTTGGGAGGGTGGG-3′

Bold letters represent Trichophaea saccata enzyme coding sequence.Restriction sites are underlined. The sequence to the left of therestriction sites is homologous to the insertion sites of pDau109 (WO2005/042735).

Extensor HIFI PCR mix, 2× concentration (Thermo Scientific cat noAB-0795) was used for experiment.

The amplification reaction (25 μl) was performed according to themanufacturer's instructions (Thermo Scientific cat no AB-0795) with thefollowing final concentrations:

PCR mix:

0.5 μM Primer F-80470

0.5 μM Primer R-80470

12.5 μl Extensor HIFI PCR mix, 2×conc.

11.0 μl H2O

10 ng of Trichophaea saccata CBS804.70 genomic DNA.

The PCR reaction was incubated in a DYAD® Dual-Block Thermal Cycler(BioRad, USA) programmed for 1 cycle at 94° C. for 30 seconds; 30 cycleseach at 94° C. for 30 seconds, 52° C. for 30 seconds and 68° C. for 60seconds followed by 1 cycle at 68° C. for 6 minutes. Samples were cooledto 10° C. before removal and further processing.

Three μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mMdisodium EDTA (TAE) buffer. A major band of about 946 bp was observed.The remaining PCR reaction was purified directly with an ILLUSTRA™ GFX™PCR DNA and Gel Band Purification Kit (GE Healthcare, Piscataway, N.J.,USA) according to the manufacturer's instructions.

Two μg of plasmid pDau109 was digested with Bam HI and Hind III and thedigested plasmid was run on a 1% agarose gel using 50 mM Tris base-50 mMboric acid-1 mM disodium EDTA (TBE) buffer in order to remove thestuffer fragment from the restricted plasmid. The bands were visualizedby the addition of SYBR® Safe DNA gel stain (Life TechnologiesCorporation, Grand Island, N.Y., USA) and use of a 470 nm wavelengthtransilluminator. The band corresponding to the restricted plasmid wasexcised and purified using an ILLUSTRA™ GFX™ PCR DNA and Gel BandPurification Kit. The plasmid was eluted into 10 mM Tris pH 8.0 and itsconcentration adjusted to 20 ng per μl. An IN-FUSION® PCR Cloning Kit(Clontech Laboratories, Inc., Mountain View, Calif., USA) was used toclone the 983 bp PCR fragment into pDau109 digested with Bam HI and HindIII (20 ng). The IN-FUSION® total reaction volume was 10 μl. TheIN-FUSION® total reaction volume was 10 μl. The IN-FUSION® reaction wastransformed into FUSION-BLUE™ E. coli cells (Clontech Laboratories,Inc., Mountain View, Calif., USA) according to the manufacturer'sprotocol and plated onto LB agar plates supplemented with 50 μg ofampicillin per ml. After incubation overnight at 37° C., transformantcolonies were observed growing under selection on the LB platessupplemented with 50 μg of ampicillin per ml.

Several colonies were selected for analysis by colony PCR using thepDau109 vector primers described below. Four colonies were transferredfrom the LB plates supplemented with 50 μg of ampicillin per ml with ayellow inoculation pin (Nunc A/S, Denmark) to new LB plates supplementedwith 50 μg of ampicillin per ml and incubated overnight at 37° C.

Primer 8653: (SEQ ID NO: 8) 5′-GCAAGGGATGCCATGCTTGG-3′ Primer 8654:(SEQ ID NO: 9) 5′-CATATAACCAATTGCCCTC-3′

Each of the three colonies were transferred directly into 200 μl PCRtubes composed of 5 μl of 2× Extensor HIFI PCR mix, (Thermo FisherScientific, Rockford, Ill., USA), 0.5 μl of primer 8653 (10 pm/μl), 0.5μl of primer 8654 (10 pm/μl), and 4 μl of deionized water. Each colonyPCR was incubated in a DYAD® Dual-Block Thermal Cycler programmed for 1cycle at 94° C. for 60 seconds; 30 cycles each at 95° C. for 30 seconds,60° C. for 45 seconds, 72° C. for 60 seconds, 68° C. for 10 minutes, and10° C. for 10 minutes.

Three μl of each completed PCR reaction were submitted to 1% agarose gelelectrophoresis using TAE buffer. All four E. coli transformants showeda PCR band of about 980 bp. Plasmid DNA was isolated from each of thefour colonies using a QIAprep Spin Miniprep Kit (QIAGEN GMBH, HildenGermany). The resulting plasmid DNA was sequenced with primers 8653 and8654 (SEQ ID NO: 8 and 9) using an Applied Biosystems Model 3730Automated DNA Sequencer using version 3.1 BIG-DYE™ terminator chemistry(Applied Biosystems, Inc., Foster City, Calif., USA). One plasmid,designated pKKSC0312-2, was chosen for transforming Aspergillus oryzaeMT3568. A. oryzae MT3568 is an amdS (acetamidase) disrupted genederivative of Aspergillus oryzae JaL355 (WO 2002/40694) in which pyrGauxotrophy was restored by inactivating the A. oryzae amdS gene.Protoplasts of A. oryzae MT3568 were prepared according to the methoddescribed in European Patent, EP0238023, pages 14-15.

E. coli 3701 containing pKKSC0312-2 was grown overnight according to themanufacturer's instructions (Genomed) and plasmid DNA of pKKSC0312-2 wasisolated using a Plasmid Midi Kit (Genomed JETquick kit, cat.nr. 400250,GENOMED GmbH, Germany) according to the manufacturer's instructions. Thepurified plasmid DNA was transformed into Aspergillus oryzae MT3568. A.oryzae MT3568 protoplasts were prepared according to the method ofChristensen et al., 1988, Bio/Technology 6: 1419-1422. The selectionplates consisted of COVE sucrose with +10 mM acetamide +15 mMCsCl+TRITON® X-100 (50 μl/500 ml). The plates were incubated at 37° C.Briefly, 8 μl of plasmid DNA representing 3 ugs of DNA was added to 100μl MT3568 protoplasts. 250 μl of 60% PEG solution was added and thetubes were gently mixed and incubate at 37° for 30 minutes. The mix wasadded to 10 ml of pre melted Cove top agarose (The top agarose meltedand then the temperature equilibrated to 40 C in a warm water bathbefore being added to the protoplast mixture). The combined mixture wasthen plated on two Cove-sucrose selection petri plates with 10 mMAcetamide. The plates were incubated at 37° C. for 4 days. SingleAspergillus transformed colonies were identified by growth on platesusing the selection Acetimide as a carbon source. Each of the four A.oryzae transformants were inoculated into 750 μl of YP mediumsupplemented with 2% glucose and also 750 μl of 2% maltodextrin and alsoDAP4C in 96 well deep plates and incubated at 37° C. stationary for 4days. At the same time the four transformants were restreaked on COVE-2sucrose agar medium.

Culture broth from the Aspergillus oryzae transformants were thenanalyzed for production of the GH24 polypeptide by SDS-PAGE usingNUPAGE® 10% Bis-Tris SDS gels (Invitrogen, Carlsbad, Calif., USA)according to the manufacturer's recommendations. A protein band atapproximately 27 kDa was observed for each of the Aspergillus oryzaetransformants. One A. oryzae transformant was cultivated in 1000 mlErlenmeyer shake flasks containing 100 ml of DAP4C medium at 26° C. for4 days with agitation at 85 rpm.

Example 3: Purification of the GH24 Lysozyme from Trichophaea saccata

The fermentation supernatant with the GH24 lysozyme from example 3 wasfiltered through a Fast PES Bottle top filter with a 0.22 μm cut-off.The resulting solution was diafiltrated with 5 mM Na-acetate, pH 4.5 andconcentrated (volume reduced by a factor of 10) on an Ultra FiltrationUnit (Sartorius) with a 10 kDa cut-off membrane.

After pretreatment about 275 mL of the lysozyme containing solution waspurified by chromatography on SP Sepharose (approximately 60 mL) in aXK26 column eluting the bound lysozyme with 0 to 100% gradient of bufferA (50 mM Na-acetate pH 4.5) and buffer B (50 mM Na-acetate+1 M NaCl pH4.5) over 10 column volumes. The fractions from the column were pooledbased on the chromatogram (absorption at 280 and 254 nm) and SDS-PAGEanalysis.

The molecular weight, as estimated from SDS-PAGE, was approximately 27kDa and the purity was >90%.

Example 4: Other Characteristics for the GH24 Lysozyme from Trichophaeasaccata

Determination of the N-terminal sequence was: YPVKTDL.

The calculated molecular weight from this mature sequence is 26205.5 Da(M+H)⁺.

The molecular weight determined by intact molecular weight analysis was26205.3 Da. (M+H)⁺.

The mature sequence (from EDMAN N-terminal sequencing data, intactmolecular weight analysis and proteomic analysis):

(SEQ ID NO: 4) YPVKTDLHCRSSPSTSASIVRTYSSGTEVQIQCQTTGTSVQGSNVWDKTQHGCYVADYYVKTGHSGIFTTKCGSSSGGGSCKPPPINAATVALIKEFEGFVPKPAPDPIGLPTVGYGHLCKTKGCKEVPYSFPLTQETATKLLQSDIKTFTSCVSNYVKDSVKLNDNQYGALASWAFNVGCGNVQTSSLIKRLNAGENPNTVAAQELPKWKYAGGKVMPGLVRRRNAEVALFKKPSSVQA HPPKC.

Example 5: Determination of Lysozyme Activity

Lysozyme activity was determined by measuring the decrease (drop) inabsorbance/optical density of a solution of resuspended Micrococcuslysodeikticus ATTC No. 4698 (Sigma-Aldrich M3770) or Exiguobacteriumundea (DSM14481) measured in a spectrophotometer at 540 nm.

Preparation of Micrococcus lysodeikticus Substrate

Before use the cells were resuspended in citric acid—phosphate buffer pH6.5 to a concentration of 0.5 mg cells/mL and the optical density (OD)at 540 nm was measured. The cell suspension was then adjusted so thatthe cell concentration equalled an OD540=1.0. The adjusted cellsuspension was then stored cold before use. Resuspended cells were usedwithin 4 hours.

Preparation of Dried Cells of Exiguobacterium undae Substrate

A culture of E. undae (DSM14481) was grown in 100 mL LB medium (Fluka51208, 25 g/L) in a 500 mL shake-flask at 30° C., 250 rpm overnight. Theovernight culture was then centrifuged at 20° C. and 5000 g for 10minutes, and the pellet was then washed twice with sterile milliQ water,and resuspended in Milli-Q water. The washed cells were centrifuged for1 minute at 13000 rpm and as much as possible of the supernatant wasdecanted. The washed cells were dried in a vacuum centrifuge for 1 hour.The cell pellet was resuspended in citric acid—phosphate buffer pH 4, 5or 6 so that the optical density (OD) at 540 nm=1.

Measurement of Lysozyme Antimicrobial Activity in the Turbidity Assay

The lysozyme sample to be measured was diluted to a concentration of100-200 mg enzyme protein/L in citric acid—phosphate buffer pH 4, 5 or6, and kept on ice until use. In a 96 well microtiterplate (Nunc) 200 μLof the substrate was added to each well, and the plate was incubated at37° C. for 5 minutes in a VERSAmax microplate reader (MolecularDevices). Following incubation, the absorbance of each well was measuredat 540 nm (start value). To start the activity measurement, 20 μL of thediluted lysozyme sample was added to each substrate (200 μL) and kineticmeasurement of absorbance at 540 nm was initiated for minimum 30 minutesup to 24 hours at 37° C. The measured absorbance at 540 nm was monitoredfor each well and over time a drop in absorbance is seen if the lysozymehas lysozyme activity. The results are presented in table 2 below.

TABLE 2 Lysozyme Activity against Micrococcus lysodeikticus andExiguobacterium undea as measured by Optical Density Drop MicrococcusExiguobacterium Lysozyme lysodeikticus ¹ undae ¹ GH22 lysozyme from +++(pH 6) + (pH 6) Gallus gallus (SEQ ID NO: 5) GH24 lysozyme from  ++ (pH6) ++ (pH 6)  Trichophaea saccate (SEQ ID NO: 4) GH25 lysozyme from   +(pH 4) + (pH 5) A. alcalophilum (SEQ ID NO: 1) ¹− Means no effect; +means small effect; ++ means medium effect; +++ means large effect. ThepH value in the brackets lists the assay pH based on lysozyme-substratecombination.

The data confirms that the GH22 lysozyme from Gallus gallus, the GH24lysozyme from Trichophaea saccata and the GH25 lysozyme from A.alcalophilum all have lysozyme activity.

Example 6: Determination of Antimicrobial Activity

The antimicrobial activity of the GH25 lysozyme from Aspergillusfumigatus (SEQ ID NO: 1), the GH24 lysozyme from Trichophaea saccata(SEQ ID NO: 4) and the GH22 lysozyme from Gallus gallus (Hen Egg Whitelysozyme (HEWL), Sigma, 62971, SEQ ID NO: 5) against Clostridiumperfringens DSM756 was tested using an RDA as described previously byLehrer et al. (Lehrer R I, Rosenman M, Harwig S S et al. (1991),“Ultrasensitive assays for endogenous antimicrobial polypeptides”, JImmunol Methods, 137:167-73), but with several modifications.

Briefly, RDA bacteria were prepared by streaking C. perfringens DSM756from freeze stocks on Luria-Bertani agar plates (Sigma L3027) and theplates were incubated overnight at 37° C. under anaerobic conditions(Anaerogen, Oxoid) in a jar. The following day colonies were suspendedin 0.9% NaCl and the suspensions were adjusted to McFarland std. 1. 87%sterile glycerol was added to give a final glycerol concentration of 20%and the cells were frozen at −80° C. until use. For estimation of colonyforming units (CFU) per milliliter of the RDA bacteria 10-fold dilutionseries were prepared of the freeze stock in 0.9% NaCl and 100 μl of thedilutions were plated on Luria-Bertani agar plates (Sigma L3027) andincubated overnight at 37° C. under anaerobic conditions (Anaerogen,Oxoid) in a jar.

When preparing the RDA plates broth media with agar was melted andcooled to 42° C. Two media's were tested in the experiment:

-   -   a) ½ Mueller-Hinton broth (MHB) (Sigma/Fluka, 90922) (i.e.        adjusted to pH6 with 4M HCl and diluted 1:1 with water) with        1.5% agarose, and    -   b) 1/10 Mueller-Hinton broth (MHB) (Sigma/Fluka, 90922) (i.e.        diluted 1:9 with water) with 1% agarose.

For each assay plate 30 ml of melted media was added to achieve around5.0×10⁵ cfu/mL C. perfringens DSM756 and this was poured into asingle-well omnitray (Nunc) plate. The omnitray plate was overlaid witha TSP plate (Nunc) and left to solidify (at room temperature or below).Afterwards, the TSP plate was removed; leaving 96 wells, in which 10 μLof the compound of interest could be tested.

10 μl of the test solutions were spotted pr. well and the plates wereincubated over night at 37° C. in a jar under anaerobic condition(Anaerogen, Oxoid). The following day a clearing zone indicatedinhibition of growth of test bacteria and thereby antimicrobialactivity. For the RDA plates with ½ MHB, the clearing zones werevisualized by coloring with MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellowtertrazole), that is reduced to purple formazan in living cells(Mosmann, Tim (1983), “Rapid colorimetric assay for cellular growth andsurvival: application to proliferation and cytotoxicity assays”, Journalof Immunological Methods 65 (1-2): 55-63). This coloring provides for adark coloring of living cells and no coloring of the clearing zoneswithout living cells.

Bacitracin zinc salt (Sigma B-8800) (50 μg/ml) was included as apositive control and lysozymes were tested using a solution of 100μg/ml. The results are presented in table 3 below.

TABLE 3 Antimicrobial Activity against Clostridium perfringens asmeasure perfringensd by RDA Diameter of clearing zone (mm) Experiment 12 1 2 Lysozyme 1/10 MHB 1/10 MHB 1/2 MHB 1/2 MHB pH 7 pH 7 pH 6 pH 6GH25 lysozyme from 13 11 6(20*) 4(19*) A. alcalophilum (SEQ ID NO: 1)GH24 lysozyme from 9 8 11 10 Trichophaea saccata (SEQ ID NO: 4) GH22lysozyme from 11 9 0 0 Gallus gallus (SEQ ID NO: 5) Bacitracin zinc salt23 20 13 11 *Incomplete inhibition of growth visible after MTT coloring

Both the GH24 lysozyme from Trichophaea saccata and the GH25 lysozymefrom Acremonium alcalophilum showed antimicrobial activity againstviable cells of C. perfringens DSM756 under both conditions tested. Forthe GH24 lysozyme from Trichophaea saccata zones of around 8-11 mm werepresent around the wells challenged with the enzyme. For the GH25lysozyme from Acremonium alcalophilum a zone of 11-13 mm was present in1/10 MBH, pH7, while in ½ MHB, pH 6 both a smaller clearing zone with adiameter of 4-6 mm and a larger incomplete clearing zone of 19-20 mmwere present around the wells challenged with the enzyme. The largerincomplete clearing zone became present after coloring with MTT. ForHEWL no zone of inhibition was observed in ½ MHB, pH 6, while in 1/10MHB, pH7 growth inhibition was observed resulting in inhibition zones of9-11 mm.

In conclusion, whilst HEWL only inhibitied growth in 1/10 MHB pH7, theGH24 lysozyme and the GH25 lysozyme surprisingly inhibitied growth of C.perfringens DSM756 under both sets of conditions.

Example 7: In Vivo Broiler Trial 1 Materials and Methods

The trial was performed at the Research Center for Animal Nutrition (DSMNutritional Products France, F-68305 Village-Neuf) according to theofficial French guidelines for experiments with live animals. Day-oldmale broiler chickens (“ROSS PM3”), were supplied by a commercialhatchery (Joseph Grelier S. A., Elevage avicole de la Bohadière, F-49290Saint-Laurent de la Plaine, France).

Animals and Housing

On the day of arrival (day 1), the chickens were divided by weight intogroups of 20 birds. Each group was placed in one floor-pen littered withwood shavings and allocated to one of the different treatments.

Each treatment was replicated with 8 groups. The chickens were housed inan environmentally controlled room. The room temperature was adapted tothe age of the birds. In the first few days an additional infra-redelectric heating lamp was placed in each pen. Moreover, in the firstweek feed was offered to the birds as crumbled pellets, afterwards aspelleted feed. The birds had free access to feed and water.

Feeding and Treatments

The experimental diets (Starter and Grower) were based on soybean meal,wheat and rye (12%) as main ingredients (Table 4). The diets wereformulated to contain 222 g crude protein and 12.5 MJ/kg ME_(N) for thestarter period and 204 g crude protein and 12.7 MJ/kg ME_(N) for thegrower period. The basal diets did not contain any coccidiostat.

TABLE 4 Composition and nutrient contents of the basal experimentaldiets Starter (d 1-22) Grower (d 22-36) Ingredients (%) Soybean meal37.65 32.80 Corn 22.35 23.05 Wheat 20.00 24.20 Rye 12.00 12.00 Soya oil3.90 4.00 DL-Methionine 0.20 0.10 L-Lysine — — NaCl 0.20 0.15 DCP 1.801.80 CaCO3 0.90 0.90 Premix¹ 1.00 1.00 Calculated content Crude protein(%) 22.2 20.4 Metabolizable energy (MJ/kg)² 12.5 12.6 Analyzed contentCrude protein (%) 22.4 20.1 Metabolizable energy (MJ/kg)³ 12.8 12.7¹Vitamin-mineral premix provided per kilogram of diet: Vitamin A: 10'000I.U.; vitamin E: 40 I.U.; vitamin K3: 3.0 mg; vitamin C: 100 mg; vitaminB1: 2.50 mg; vitamin B2: 8.00 mg; vitamin B6: 5.00 mg; vitamin B12: 0.03mg; niacin: 50.0 mg; pantothenate calcium: 12.0 mg; folic acid: 1.50 mg;biotin 0.15 mg; cholin: 450 mg; ethoxyquine: 54 mg; Na: 1.17 g; Mg: 0.8g; Mn: 80 mg; Fe: 60 mg; Cu: 30 mg; Zn: 54 mg; I: 1.24 mg; Co: 0.6 mg;Se: 0.3 mg ¹Without coccidiostat; ²Calculated with EC-equation;³Calculated with EC-equation based on analysed crude nutrients

The diets were fed either unsupplemented (negative control, C),supplemented with the GH25 lysozyme (SEQ ID NO: 1) at 25, 50, 100, or200 mg per kg feed or supplemented with Avilamycin at an inclusion levelof 10 mg/kg as positive control. No additional enzymes (e.g. phytase)were added to the feed.

Appropriate amounts of the solid product (Avilamycin) was mixed with asmall quantity of the basal feed as a premix which was then added to thefeed to get the final concentration, according to the treatment. Aftermixing the feed was pelleted (3×25 mm) at about 70° C.

Appropriate amount of the liquid preparations of Lysozyme was diluted inwater and sprayed onto the respective pelleted feed to get the finalconcentrations in the feed corresponding to the different treatments.For procedural balance of all treatments the same volume of water werealso sprayed onto the pellets of the control diets

Experimental Parameters and Analyses

For the two experiments, the birds were weighed (as replicate group) ondays 1, 22 and 36. The feed consumption for the intermediate periods wasdetermined. Body weight gain and feed conversion ratio (feed/gain) werecalculated.

The analyses of the nutrient content in the feed samples were performedaccording to standard methods (VDLUFA 1976). Nitrogen analysis wascarried out with a Leco N analyzer (CP=N*6.25).

Statistical Analysis

For the statistical evaluation of performance data, a one-factorialanalysis of variance (factor: treatment) was carried out. The software‘Stet Box Pro Agri’, version 7.1.9 (Grimmer soft, 1985-2011) was used.Where significant treatment effects (p<0.05) were indicated, thedifferences among treatment means were subsequently determined with theNewman-Keuls test.

Results and Discussion

Based on the proximate chemical analyses in the diets the content ofcrude protein and metabolizable energy was close to the calculatedcontent for both the starter and the grower diets (Table 4).

The results of the growth performance are summarized in table 5 for thetwo periods (starter period, day 1-22; grower period, day 22-36) and forthe whole experimental period from day 1 to day 36.

During the starter period, although not significant, the supplementationof graded inclusion levels of microbial lysozyme was effective inimproving the weight gain (WG), compared to the control diet. Anumerical improvement by 1.3%, 2.4% and 4.2% was recorded with 25, 100and 200 mg/kg of lysozyme, respectively. Moreover, the inclusion of 200mg lysozyme resulted in comparable improvement of the WG as theinclusion of 10 mg/kg Avilamycin (+4.1%).

Lysozyme supplementation led to a significant improvement of the feedconversion ratio (FCR) compared to the control diet. The FCR wasimproved by 2.0%, 5.1% and 6.6% with the addition of 25, 100 and 200mg/kg of lysozyme, respectively. The FCR was significantly differentbetween the treatments supplemented with 25 and 200 mg/kg lysozyme. Theinclusion of 100 and 200 mg/kg of lysozyme resulted in similar effect onFCR as the inclusion of 10 mg/kg Avilamycin.

The inclusion of the lysozyme at 25, 50 and 100 mg/kg led to asignificant improvement of the FCR by 3.3%, 2.9% and 3.2%, respectively,compared to the control diet. The addition of Avilamycin resulted in anumerical improvement of the FCR by 2.3%, compared to the control diet.For the overall trial period from day 1 to day 36, lysozymesupplementation was not effective in improving the WG, although apositive trend (+1.6%) was recorded with the addition of 25 mg/kglysozyme, compared to the control diet. A numerical improvement by 1.5%was recorded with the addition of 10 mg/kg Avilamycin. The feed intakewas significantly affected with the addition of 100 mg/kg lysozymecompared to the control diet. However, the differences among thesupplemented treatments were not significant.

The FCR was significantly improved with lysozyme and Avilamycinsupplementation compared to the control diet. An improvement in a rangeof 2.1% to 4.4% was obtained with graded inclusion levels of lysozyme.Moreover, the inclusion of lysozyme at 100 mg/kg resulted in asignificant better improvement of FCR compared to the inclusion of 200mg/kg. Avilamycin supplementation led to a significant improvement by 4%compared to the control diet.

TABLE 5 Growth performance data¹ of male broiler chickens fed gradedinclusion levels of microbial lysozyme Product Starter (d 1-22) Grower(d 22-36) Whole period Weight Feed Weight Feed Weight Feed gain intakegain intake gain intake Treatment (g/b) (g/b) FCR (g/b) (g/b) FCR (g/b)(g/b) FCR Control (C) 1101 1995 1.816^(a) 1760 3049^(a) 1.734^(ab) 28615047^(a) 1.764^(a) Avilamycin 1147 1943 1.695^(c) 1757 2976^(ab)1.694^(abc) 2903 4918^(ab) 1.694^(bc) (10 mg/kg) Relative to C (%) 104.197.4 93.3 99.8  97.6 97.7 101.5  97.5 96 SEQ ID NO: 1 1115 19851.781^(ab) 1792 3004^(ab) 1.676^(c) 2908 4991^(ab) 1.716^(bc) (25 mg/kg)Relative to C (%) 101.3 99.5 98 101.9  98.5 96.7 101.6  98.9 97.3 SEQ IDNO: 1 1102 1977 1.793^(ab) 1747 2939^(ab) 1.683^(bc) 2849 4920^(ab)1.727^(b) (50 mg/kg) Relative to C (%) 100.1 99.1 98.7 99.3  96.4 97.199.6  97.5 97.9 SEQ ID NO: 1 1127 1941 1.723^(bc) 1729 2870^(b)1.662^(c) 2856 4815^(b) 1.686^(c) (100 mg/kg) Relative to C (%) 102.497.3 94.9 98.2  94.1 95.8 99.8  95.4 95.6 SEQ ID NO: 1 1148 19461.696^(c) 1685 2934^(ab) 1.742^(a) 2832 4877^(ab) 1.722^(b) (200 mg/kg)Relative to C (%) 104.2 97.6 93.4 95.7  96.2 100.5 99  96.6 97.6 Pvalues 0.269 0.603 <0.001 0.065   0.028 0.001 0.273   0.017 <0.001Newman-Keuls test: Means within a row, not sharing a common superscript,are significantly different (p < 0.05). ¹The performance were calculatedwith n = 8 groups of 20 birds per treatment.

Conclusion

The results obtained in the study showed that the inclusion of microbiallysozyme was effective in improving the feed conversion ratio ofbroilers fed diets formulated without coccidiostat and based on soybeanmeal, wheat and rye. Even at lower dosage (25 mg/kg) microbial lysozymesupplementation showed significant improvement of the FCR compared tothe control diet. Moreover, the effects were comparable to theseobtained with the inclusion of Avilamycin at 10 mg/kg.

Example 8: In Vivo Broiler Trial 2

The in vivo trial was carried out as described in example 7, except thefeed was supplemented with the GH25 lysozyme (SEQ ID NO: 1) at 6.25,12.5 or 25 mg per kg feed or supplemented with Avilamycin at aninclusion level of 10 mg/kg. No additional enzymes (e.g. phytase) wereadded to the feed.

The experimental diets (Starter and Grower) were based on soybean meal,wheat and rye (12%) as main ingredients (Table 6). The diets wereformulated to contain 222 g crude protein and 12.5 MJ/kg ME_(N) for thestarter period and 204 g crude protein and 12.7 MJ/kg ME_(N) for thegrower period. The basal diets did not contain any coccidiostat.

TABLE 6 Composition and nutrient contents of the basal experimentaldiets Starter (d 1-22) Grower (d 22-36) Ingredients (%) Soybean meal34.70 30.00 Corn 25.45 25.55 Wheat 20.00 25.00 Rye 12.00 12.00 Soya oil3.70 3.50 DL-Methionine 0.20 0.10 L-Lysine 0.05 — NaCl 0.20 0.15 DCP1.80 1.80 CaCO3 0.90 0.90 Premix¹ 1.00 1.00 Calculated content Crudeprotein (%) 22.3 20.5 Metabolizable energy (MJ/kg)² 12.5 12.6 Analyzedcontent Crude protein (%) 22.3 20.3 Metabolizable energy (MJ/kg)³ 12.812.5 ¹Vitamin-mineral premix provided per kilogram of diet: Vitamin A:10'000 I.U.; vitamin E: 40 I.U.; vitamin K3: 3.0 mg; vitamin C: 100 mg;vitamin B1: 2.50 mg; vitamin B2: 8.00 mg; vitamin B6: 5.00 mg; vitaminB12: 0.03 mg; niacin: 50.0 mg; pantothenate calcium: 12.0 mg; folicacid: 1.50 mg; biotin 0.15 mg; cholin: 450 mg; ethoxyquine: 54 mg; Na:1.17 g; Mg: 0.8 g; Mn: 80 mg; Fe: 60 mg; Cu: 30 mg; Zn: 54 mg; I: 1.24mg; Co: 0.6 mg; Se: 0.3 mg ¹Without coccidiostat; ²Calculated withEC-equation; ³Calculated with EC-equation based on analysed crudenutrients

Results and Discussion

Based on the proximate chemical analyses in the diets the content ofcrude protein and metabolizable energy was close to the calculatedcontent for both the starter and the grower diets (Table 6).

The results of the growth performance are summarized in table 7 for thetwo periods (starter period, day 1-22; grower period, day 22-36) and forthe whole experimental period from day 1 to day 36.

During the starter period, the supplementation of the lysozyme was noteffective in improving significantly the weight gain (WG), compared tothe control diet (C). However, a numerical improvement by 3.6% wasrecorded with the supplementation of lysozyme at 6.25 mg/kg. Theinclusion of 12.5 and 25 mg/kg of lysozyme resulted in significantlybetter effect on FCR than the inclusion of 10 mg/kg Avilamycin. The FCRwas numerically improved by 2.9% and 3.4% with the addition of 12.5 and25 mg/kg lysozyme compared to the control diet. No improvement of the WGand the FCR was obtained with Avilamycin supplementation.

In the grower period, the supplementation of the lysozyme was noteffective in improving significantly the WG and the FCR, compared to thecontrol diet. Only numerical improvement by 2.9 and 2.0% was recordedfor the WG and the FCR, respectively, with the supplementation oflysozyme at 25 mg/kg compared to the control diet. During this period,Avilamycin supplementation resulted in numerical improvement of the WGby 1.2%, whereas a significant improvement of the FCR by 5.8%, wasrecorded compared to the control diet.

For the overall trial period from day 1 to day 36, supplementation ofthe lysozyme was effective in improving the WG, compared to C, atinclusion level of 25 mg/kg (+2.0%). The FCR was significantly improvedwith lysozyme (2.2%) at 25 mg/kg and Avilamycin (2.6%) supplementationcompared to the control diet. Further, a numerical improvement in theFCR of 1.9% was obtained using lysozyme at 12.5 mg/kg.

TABLE 7 Growth performance data¹ of male broiler chickens fed gradedinclusion levels of microbial lysozyme Product Starter (d 1-22) Grower(d 22-36) Whole period Weight Feed Weight Feed Weight Feed gain intakegain intake gain intake Treatment (g/b) (g/b) FCR (g/b) (g/b) FCR (g/b)(g/b) FCR Control (C) 1067 1471^(bcd) 1.379^(ab) 1586 2730 1.721^(a)2653 4188 1.578^(ab) Avilamycin 1070 1517^(a) 1.421^(a) 1605 25991.621^(b) 2675 4111 1.537^(c) (10 mg/kg) Relative to C (%) 100.2  103.1103 101.2 95.2 94.2 100.8 98.1 97.4 SEQ ID NO: 1 1106 1551^(ab)1.403^(ab) 1545 2705 1.753^(a) 2651 4249 1.604^(a) (6.25 mg/kg) Relativeto C (%) 103.6  105.4 101.8 97.4 99.1 101.9 99.9 101.4 101.6 SEQ ID NO:1 1049 1403^(d) 1.339^(b) 1581 2671 1.692^(a) 2630 4069 1.548^(bc) (12.5mg/kg) Relative to C (%) 98.3  95.4 97.1 99.7 97.8 98.3 99.1 97.1 98.1SEQ ID NO: 1 1076 1434^(cd) 1.332^(b) 1633 2753 1.686^(a) 2709 41811.543^(c) (25 mg/kg) Relative to C (%) 100.9  97.4 96.6 102.9 100.8 98102.1 99.8 97.8 P values 0.205  <0.001 0.002 0.303 0.131 0.001 0.690.231 <0.001 Newman-Keuls test: Means within a row, not sharing a commonsuperscript, are significantly different (p < 0.05). ¹The performancewere calculated with n = 8 groups of 20 birds per treatment.

Conclusion

The results obtained in the study showed that the inclusion of amicrobial lysozyme was effective in improving the feed conversion ratioof broilers fed diets formulated without coccidiostat and based onsoybean meal, wheat and rye. At 25 mg/kg, the microbial lysozymesupplementation showed significant improvement of the FCR compared tothe control diet and even at 12.5 mg/kg, the microbial lysozyme showednumeral improvements. Moreover, the effects were comparable to theseobtained with the inclusion of Avilamycin at 10 mg/kg.

Example 9: In Vivo Broiler Trial 3

The in vivo trial was carried out as described in example 7, except thefeed was supplemented with the GH25 lysozyme (SEQ ID NO: 1) at 12.5 or25 mg per kg feed, the GH24 lysozyme (SEQ ID NO: 4) at 12.5 or 25 mg perkg feed or with Avilamycin at an inclusion level of 10 mg/kg. Noadditional enzymes (e.g. phytase) were added to the feed.

The experimental diets (Starter, Grower and Finisher) were based onsoybean meal, wheat and rye (12%) as main ingredients (Table 8). Thediets were formulated to contain 225 g crude protein and 12.5 MJ/kgME_(N) for the starter period, 215 g crude protein and 12.8 MJ/kg ME_(N)for the grower period and 205 g crude protein and 13.0 MJ/kg ME_(N) forthe finisher period.

TABLE 8 Composition and nutrient contents of the basal experimentaldiets Starter Grower Finisher (d 1-22) (d 22-36) (d 36-42) Ingredients(%) Soybean meal 38.00 35.40 33.00 Corn 22.55 20.20 21.40 Wheat 20.0024.50 25.00 Rye 12.00 12.00 12.00 Soya oil 3.80 4.30 4.85 DL-Methionine0.20 0.15 0.15 NaCl 0.15 0.15 0.15 DCP 1.70 1.85 1.95 CaCO3 0.54 0.390.34 Premix¹ 1.00 1.00 1.00 Coccidiostat 0.06 — — Calculated contentCrude protein (%) 22.5 21.5 20.5 Metabolizable energy 12.6 12.8 13.0(MJ/kg)² Analyzed content Crude protein (%) 21.9 21.5 20.8 Metabolizableenergy 12.5 12.6 12.9 (MJ/kg)³ ¹Vitamin-mineral premix provided perkilogram of diet: Vitamin A: 10'000 I.U.; vitamin E: 40 I.U.; vitaminK3: 3.0 mg; vitamin C: 100 mg; vitamin B1: 2.50 mg; vitamin B2: 8.00 mg;vitamin B6: 5.00 mg; vitamin B12: 0.03 mg; niacin: 50.0 mg; pantothenatecalcium: 12.0 mg; folic acid: 1.50 mg; biotin 0.15 mg; cholin: 450 mg;ethoxyquine: 54 mg; Na: 1.17 g; Mg: 0.8 g; Mn: 80 mg; Fe: 60 mg; Cu: 30mg; Zn: 54 mg; I: 1.24 mg; Co: 0.6 mg; Se: 0.3 mg. ¹Withoutcoccidiostat; ²Calculated with EC-equation; ³Calculated with EC-equationbased on analysed crude nutrients.

Results and Discussion

Based on the analyzed chemical compositions of the diets, the content ofcrude protein was close to the calculated content but the metabolizableenergy was higher than expected in all the three diets (starter-growerand finisher) (Table 8).

The results of the growth performance are summarized in table 9 for thetwo periods (starter period, day 1-22; grower period, day 22-36,finisher period, day 36-42) and for the whole experimental period fromday 1 to day 42 (table 10).

TABLE 9 Growth performance data of male broiler chickens fed gradedinclusion levels of microbial lysozyme Product Starter (d 1-22) Grower(d 22-36) Finisher (d 36-42) Weight Feed Weight Feed Weight Feed gainintake gain intake gain intake Treatment (g/b) (g/b) FCR (g/b) (g/b) FCR(g/b) (g/b) FCR Control (C) 1096 1578 1.440 1328 2285 1.723 761 14871.960 Avilamycin 1102 1593 1.452 1334 2242 1.683 738 1460 1.982 (10mg/kg) Relative to C (%) 100.6 101.0 100.8 100.4 98.1 97.7 97.0 98.2101.1 SEQ ID NO: 1 1106 1621 1.466 1365 2365 1.734 794 1544 1.953 (12.5mg/kg) Relative to C (%) 100.9 102.7 101.8 102.8 103.5 100.6 104.2 103.899.6 SEQ ID NO: 1 1129 1596 1.413 1364 2308 1.692 775 1522 1.968 (25mg/kg) Relative to C (%) 103.0 101.1 98.1 102.7 101.0 98.2 101.8 102.3100.4 SEQ ID NO: 4 1095 1608 1.469 1359 2298 1.692 795 1560 1.969 (12.5mg/kg) Relative to C (%) 99.9 101.9 102.0 102.4 100.5 98.2 104.4 104.8100.4 SEQ ID NO: 4 1133 1603 1.414 1364 2336 1.714 800 1524 1.919 (25mg/kg) Relative to C (%) 103.4 101.6 98.1 102.7 102.2 99.5 105.1 102.597.9

TABLE 10 Growth performance summary of male broiler chickens fed gradedinclusion levels of microbial lysozyme Whole period Treatment/ WeightFeed Product gain (g/b) intake (g/b) FCR Motality EPEF Control (C) 31865351 1.680 7.5 418 Avilamycin 3184 5312 1.669 12.5 397 (10 mg/kg)Relative to C (%) 99.9 99.3 99.3 95.0 SEQ ID NO: 1 3264 5529 1.695 13.1398 (12.5 mg/kg) Relative to C (%) 102.5 103.3 100.9 95.2 SEQ ID NO: 13269 5426 1.660 8.8 428 (25 mg/kg) Relative to C (%) 102.6 101.4 98.8102.4 SEQ ID NO: 4 3249 5466 1.683 11.3 408 (12.5 mg/kg) Relative to C(%) 102.0 102.1 100.2 97.6 SEQ ID NO: 4 3290 5450 1.657 10.0 425 (25mg/kg) Relative to C (%) 103.3 101.9 98.6 101.7

Over the whole period, the GH24 and GH25 lysozyme supplemented at 25mg/kg resulted in FCR improvements of 1.2% and 1.4% respectivelycompared to NC diet. Furthermore, the GH24 and GH25 lysozymesupplemented at 25 mg/kg resulted in EPEF improvements of 2.4% and 1.7%respectively compared to NC diet. Whilst the positive control Avilamycinshowed a slight FCR improvement of 0.7%, the EPEF was worse by 5.0%. Inthis trial, the lower supplement of 12.5 mg/kg of the GH24 and GH25lysozyme did not show any benefit over the NC diet.

Conclusion

The results obtained in the study showed that the inclusion of amicrobial lysozyme at 25 mg/kg was effective in improving the FCR andthe EPEF of broilers fed diets formulated with coccidiostat in thestarter period and based on soybean meal, corn, wheat and rye. Inaddition, the microbial lysozyme at 25 mg/kg was markedly better inimproving the EPEF over the positive control (Avilamycin).

Example 10: In Vivo Piglet Trial Materials and Methods

The trial was performed from Oct. 23 to Dec. 4, 2014 at the ResearchCenter for Animal Nutrition (DSM Nutritional Products France, F-68305Village-Neuf) according to the official French guidelines forexperiments with live animals.

Animals and Housing

One hundred and four castrated male crossbred (Large-White(female)×Redon (male)) weaned piglets having 28 days of age supplied bythe commercial farm “Elevage de la Plaine du Rhin” located in Balgau(France), were used in a 42-day experiment. The initial bodyweight ofthe piglets was 7.88±0.675 kg. They were sorted by body weight into 32groups of 3 or 4 piglets and randomly allotted to each dietarytreatment. Each group of animals was placed in one flat-deck cages andallocated to one of the different treatments.

Each treatment was replicated with 8 cages using a total of 26 animals(6 cages of 3 animals/treatment and 2 cages of 4 animals/treatment) pertreatment. Each cage had a plastic-coated welded wire floor and wasequipped with two water nipples and two stainless-steel individualisedfeeders. Animals were housed in an environmentally controlled room. Roomtemperature was initially 27° C. and was lowered weekly by about 2° C.until 21-22° C. and relative humidity percentage was 50%.

Feeding and Treatments

The experimental diets (Pre-Starter and Starter) were fed ad libitum intwo feeding phases from day 0 to 14 (phase 1, Pre-starter) and day 14 to42 (phase 2, Starter). The ingredient composition and the calculatednutrient levels of the experimental diets for phases 1 and 2 arepresented in table 11. The analysed content is presented in table 12.Both diets were formulated to meet the animals' requirements accordingNRC (2012) and were fed in pelleted form. Pelleting conditions were at70° C. for 30 seconds.

TABLE 11 Composition and nutrient contents of the basal experimentaldiets Pre-starter (%) Starter (%) Ingredients Barley 38.00 38.00 Wheat22.10 17.50 Soybean meal 48% 24.00 22.00 Maize 6.00 16.20 Soybean oil1.00 2.00 Dried whey 5.00 — Vermiculite — 1.00 Calcium carbonate 0.300.30 L-Lysine HCl 0.10 — Vitamin-mineral Premix 3136¹ 3.50 3.00Estimated nutrient content Crude protein (%) 19.47 17.95 Lysine (%) 1.241.03 Threonine (%) 0.67 0.61 Methionine + cysteine (%) 0.70 0.66 Total P(%) 0.71 0.64 Total Ca (%) 0.84 0.71 Estimated digestible energy (MJ/kg)13.62 13.87 ¹Vitamin-mineral premix 3136 provided per kilogram of diet:Vitamin A: 20'000 I.U.; Vitamin E: 100 mg.; Vitamin K: 4.0 mg; VitaminC: 200 mg; Vitamin B1: 5.00 mg; Vitamin B2: 10.00 mg; Vitamin B6: 8.00mg; Vitamin B12: 0.07 mg; Niacin: 60.0 mg; Pantothenic acid: 40.0 mg;Folic acid: 3.00 mg; Biotin 0.4 mg; Choline: 800 mg; Mn: 60.5 mg; Fe:162 mg; Cu: 9.5 mg; Zn: 100 mg; I: 0.9 mg; Se: 0.3 mg

TABLE 12 Analysed nutrient contents of the basal diets Analyzed nutrientcontent Pre-starter Starter Dry matter (%) 87.89 87.42 Crude protein (%DM) 21.69 19.63 Crude Ash (% DM) 6.41 6.37 Fat (% DM) 4.15 5.42 Starch(% DM) 44.63 49.54 Total P (mg/g DM) 8.24 7.50 Total Ca (mg/g DM) 9.017.69 Total Zn (mg/g DM) 0.28 0.25 Gross energy (MJ/kg DM) 18.42 18.67

The diets were fed either unsupplemented (negative control) orsupplemented with the GH25 lysozyme (SEQ ID NO: 1) at 50 mg per kg feed,the GH24 lysozyme (SEQ ID NO: 4) at 50 mg per kg feed or VevoVital at5000 mg per kg feed. No additional enzymes (e.g. phytase) were added tothe feed.

Treatment Product Inclusion level (mg/kg) A Negative control — BVevoVital 5000 C SEQ ID NO: 1 50 D SEQ ID NO: 4 50

VevoVital was mixed to the premixed mash diet before pelleting the diet.

Appropriate amount of the liquid preparations of lysozyme was diluted inwater and sprayed onto the respective pelleted feed to get the finalconcentrations in the feed corresponding to the different treatments.For procedural balance of all treatments the same volume of water werealso sprayed onto the pellets of the control diets.

Experimental Parameters and Analyses

The health status of the animals was controlled daily.

Body weight of the individual animals and feed consumption per pen wererecorded on days 14 and 42 of the study. Performance, average dailyweight gain (ADWG), average daily feed intake (ADFI) and feed conversionratio (FCR) was calculated for phases 1 and 2, and the wholeexperimental period.

Statistical Analysis

The experimental unit was the piglet, except for ADFI and FCR which weremeasured by cage, and in both cases, treatment was used as classvariable.

Statistical analyses were performed using the StatGraphics Centurion XVIstatistical software package (Manugistics, Rockville, Md.).

One-factorial ANOVA and Student-Newman-Keuls test was used to assessdifferences among means in treatment groups.

Variability in the data was expressed as the pooled standard error. Inall instances, differences were reported as significant at P<0.05.

Results and Discussion

Based on the analyzed chemical compositions of the diets, the content ofcrude protein (19.07% and 17.16% as is, in pre-starter and starterperiods, respectively) was close to the calculated content (19.47% and17.95% as is, in pre-starter and starter periods, respectively) (Table12).

All piglets remained healthy throughout the study. During the enzymesupplementation, none of the animals showed any symptoms of illness ortoxicosis due to the test compounds. Mortality rate was zero.

Results of the growth performance are summarized for the two periods(pre-starter period, day 0-14, and starter period, day 14-42) and forthe whole experimental periods from day 0 to day 42 (Table 12). Ingeneral, excellent animal growth performance was obtained for alltreatments.

No significant difference among the supplemented treatments was recordedin terms of body weight. However, the supplementation of either the GH25lysozyme (SEQ ID NO: 1) or the GH24 lysozyme (SEQ ID NO: 4) at 50 mg/kgresulted in a numerical improvement of the ADWG by 4.0 and 11.8%,respectively during the starter period, and 2.4% and 7.8%, respectivelyduring the whole period, compared to the negative control diet.

Over the pre-starter, starter and whole periods the supplementation ofVevoVital resulted in an improvement of ADWG by 4.8, 5.8 and 5.4%compared to NC.

The results of FCR showed a statistically significant effect (p<0.05) oftreatment in the pre-starter, starter and overall periods. Over thestarter period, from day 15 to day 42, piglets, which received VevoVital(PC), the GH25 lysozyme (SEQ ID NO: 1) or the GH24 lysozyme (SEQ ID NO:4) included at 50 mg/kg showed an improvement to FCR by 9.5, 11.3 and9.1% compared to NC.

As presented in Table 13, during the overall period (day 0 to day 42)piglets receiving feed added with VevoVital (PC) at 5000 mg/kg, the GH25lysozyme (SEQ ID NO: 1) or the GH24 lysozyme (SEQ ID NO: 4) (bothenzymes added at 50 mg/kg) showed an improvement on FCR by 9.6, 9.6 and7.1% respectively compared to the negative control.

No significant difference among the supplemented treatments was recordedin terms of EPEF. However, the supplementation of either the GH25lysozyme (SEQ ID NO: 1) or the GH24 lysozyme (SEQ ID NO: 4) at 50 mg/kgresulted in a large numerical EPEF improvement by 13.0 and 15.3%,respectively during the whole period, compared to the negative control.

TABLE 13 Growth performance data of piglets fed graded inclusion levelsof microbial lysozyme day 0-14 day 14-42 day 0-42 ADFI ADWG ADFI ADWGADFI ADWG Treatment (g/d)² (g/d)¹ FCR² (g/d)² (g/d)¹ FCR² (g/d)² (g/d)¹FCR² EPEF Negative Mean 360 290 1.240^(ab) 930^(bc) 603 1.544^(b)734^(b) 499 1.484^(b) 339 control SD 20 66 0.053  34 79 0.096  23 690.072 24 (NC) % 100 100 100 100 100 100 100 100 100 100 VevoVital Mean355 304 1.168^(a) 886^(ab) 638 1.398^(a) 709^(ab) 526 1.342^(a) 394 (PC)SD 26 56 0.041  33 62 0.103  30 52 0.079 30 % relative 98.6 104.8 94.2 95.3 105.8 90.5  96.6 105.4 90.4 116.2 NC NC+ SEQ Mean 324 2791.170^(a) 862^(a) 627 1.379^(a) 683^(a) 511 1.341^(a) 383 ID NO: 1 SD 5184 0.085  38 84 0.076  37 78 0.070 52 (50 ppm) % relative 90.0 96.2 94.4 92.7 104.0 89.3  93.1 102.4 90.4 113.0 NC NC+ SEQ Mean 333 2651.272^(b) 944^(c) 674 1.404^(a) 740^(b) 538 1.379^(a) 391 ID NO: 4 SD 4253 0.049  62 89 0.044  49 69 0.040 43 (50 ppm) % relative 92.5 91.4102.6 101.5 111.8 90.9 100.8 107.8 92.9 115.3 NC P value 0.317 0.3460.014  0.013 0.068 0.009  0.034 0.341 0.004 0.068 ¹Mean ± mean deviationof 18 determinations; ²Mean ± mean deviation of 6 determinations;^(a,b,c)Different superscripts in the same column indicate a significantdifference (p < 0.05); ADFI: average daily feed intake; ADWG: averagedaily weight gain.

Conclusion

It can be concluded that in the present study and at the tested dosagesand conditions, both lysozyme candidates supplemented at 50 mg per kgfeed to soybean meal, maize, wheat and barley based diet had anumerically improvement on growth performance of piglets although thiseffect was not statistically significant. Improvement of BWG with theGH24 lysozyme (SEQ ID NO: 4) at 50 mg/kg feed was even higher than thepositive control and there was a very large improvement of 13-15% inEPEF. Importantly, the results show a statistically significant effectof both GH25 lysozyme (SEQ ID NO: 1) and GH24 lysozyme (SEQ ID NO: 4)included at 50 mg/kg feed treatment on ADFI and FCR in the starter andoverall periods.

Example 11: In Vivo Broiler Trial 4 Treatments and Diet Composition

The basal diet was based upon wheat, rye, soybean meal, fish meal andsunflower meal, and was formulated and adjusted in two phases (Starterand Grower periods of 7 and 17 days, respectively) according to thegrowing animals changing requirements. Diet composition was designed tomeet or exceed the requirements except for metabolisable energy,phosphorus and calcium (tables 15 and 16).

Starter feed did not contain lysozyme but served for a similar rearingperiod of 7 days and introduction of the birds to the main dietcomponents in the Grower feeds. During the Grower period, the lysozyme(SEQ ID NO: 1) was applied at 50 g/t as a liquid formulation (table 14).There were no other enzymes or any coccidiostats supplemented in thediets. The diets were prepared at a feedmill specialised in experimentaldiets and the mash feed was offered ad libitum to the birds.

TABLE 14 Study design Inclusion Treatment Lysozyme level (mg/kg) PensBirds per pen 1 — — 16 40 2 SEQ ID NO: 1 50 16 40

TABLE 15 Diet composition (g/kg) Phase Starter (Days 1-7) Grower (Days8-24) Wheat 511.25 473.90 Rye 50.00 100.00 Soybean Meal (48% XP) 261.00207.00 Soybean Hulls¹ 29.00 23.00 Fishmeal 70% XP 50.00 20.00 SunflowerMeal (low XP) 70.00 Animal Fat (Lard) 30.00 Soybean Oil 62.50 42.00Premix 5.00 5.00 Lime fine 13.50 13.50 Monocalciumphosphate 9.50 4.80Salt 1.20 1.30 NaHCO3 2.10 2.60 L-Lysine HCl 1.35 2.70 DL-Methionine2.60 2.60 L-Threonine 0.95 1.15 L-Valine 0.05 0.45 ¹Soybean hulls wereintroduced to the diet in order to simulate low protein SBM, which wasnot available as a single ingredient

TABLE 16 Premix composition Supplied Nutrient premix per kg feedProvided as Vitamin A (retinyl acetate) 12,000 IE Vitamin D₃(cholecalciferol) 2,400 IE Vitamin E (dl-a-tocopherol) 50 mg Vitamin K₃(menadione) 1.5 mg Vitamin B₁ (thiamin) 2.0 mg Vitamin B₂ (riboflavin)7.5 mg Vitamin B₆ (pyridoxine-HCl) 3.5 mg Vitamin B₁₂ (cyanocobalamin)20 μg Niacin 35 mg D-pantothenic acid 10 mg Choline chloride 460 mgFolic acid 1.0 mg Biotin 0.2 mg Iron 80 mg (267 mg FeSO₄•H₂O) Copper 12mg (48 mg CuSO₄•5H₂O) Manganese 85 mg (142 mg MnO) Zinc 60 mg (169 mgZnSO₄•H₂O) Cobalt 0.40 mg (1.9 mg CoSO₄•7H₂O) Iodine 0.8 mg (1.1 mg KJ)Selenium 0.1 mg (0.22 mg Na₂SeO₃) Anti-oxidant mixture 125 mg OxytrapPXN

Animals and Housing

At the day of hatching, male day-old broiler type chickens (malebyproducts of female parental line of Cobb 500) were obtained from CobbGermany Avimex GmbH, Wiesenena (Brösenweg 80, 04509 Wiesenena).

The birds were randomly assigned in groups of 22 chickens to theexperimental pens (˜3 sq·m.) equipped with a bell drinker and a roundfeeder. After 7 days of equal rearing, number of birds per pen wasreduced to 20, selecting against obviously light birds. Recorded bodyweights (BW) were immediately statistically evaluated. In order toensure similar average BW between treatments and variation withintreatments statistical evaluation of BW-placement of chicks wascoordinated in such a way as to minimize within-pen variation andbetween-treatment differences of average BW.

Feed and water were freely available, feed consumption was recorded.Initial bedding consisted of wood shavings. Caked excreta patches aroundthe drinkers were removed several times during the experimental periodand more bedding material was added when required. Light and temperatureregimes were managed according to the breeder's recommendations.

Birds were routinely vaccinated against Newcastle disease and Gumboro onday 18.

Data Recording and Calculation of Performance Parameters

Birds were weighed (groupwise) at placement and at the end of eachfattening period. At the final weighing, birds were weighedindividually. Feed offered was recorded continuously upon refilling thefeeders; the feed remaining in the feeders was recorded at the end ofeach fattening period. From these data, feed consumption was calculated.

The weight of losses and culls was recorded upon occurrence.

Daily BW gain per bird (BW gain) and feed conversion ratio (FCR) werecalculated as follows:

BWgain: difference between BW per bird at the end and at the beginningof the study divided by the number of days

FCR: total feed consumption of a pen divided by total BW gain of thatpen (total BW gain=total BW at the end+weight of removals andlosses−total BW at the beginning) The European Production EfficacyFactor (EEF) was calculated as follows:

EEF=[(liveability,%×BWgain,kg)/(Study duration in days×FCR)]×100.

Statistical Analysis

Statistical unit was ‘pen’. Prior to statistical analysis, an outliertest (Grubb's test) was conducted. As a consequence of this procedure nodata was excluded from the dataset.

Data of performance was analysed using a bi-factorial ANOVA (procedurePROC GLM) with the fixed effects of lysozyme supplementation as well astheir interaction. Differences were investigated between the variouslevels of each main factor (Tukey test), accounting for multiplecomparisons where appropriate.

All statistical analysis was conducted using the software package SAS9.3.

Results and Discussion

Losses and culls throughout the study ranged from 0.8% to 1.6% over days8 to 24 for individual treatments. No differences between the treatmentswere detected.

TABLE 17 FCR and EPEF results using lysozyme (SEQ ID NO: 1) FCR %improve- EPEF % improve- Treatment FCR change ment EPEF change ment None1.59 — — 315 — — Lysozyme 1.56 0.03 1.9% 327 12 3.8%

The results show that there was an improvement in both FCR and EPEF whenthe GH25 lysozyme of SEQ ID NO: 1 was added to the broiler diet comparedto when no lysozyme was present.

Example 12: Microbiota Analysis from In Vivo Broiler Trial 4 (Example11)

The microbiota of broilers from in vivo broiler trial 4 (described inExamples 11) was analysed as described below. 18 chickens from treatment1 (representing four individual pens) and 30 chickens from treatment 2(representing four individual pens) were selected for analysis of themicrobiota

Sampling

At the end of the feeding trial chickens selected for microbiotaanalysis were slaughtered for collection of gut content from the twoceca. The chickens were dissected directly after slaughtering and theintestines were eviscerated. The ceca were then separated from the restof the intestines by cutting the ceca around 1 cm proximally from theileocecal junction. This was done by use of a scissor (sterilized in anethanol bath) or by use of disposable scalpels. The content of the twoceca were emptied collectively into one 15 ml tube. The content of thetube was mixed with an inoculation needle and the digesta wasdistributed into 4 separate Eppendorf tubes as small aliquots (50-500mg). The samples were snap-freezed on dry ice and placed in a −80° C.freezer until further processing.

DNA Extraction

DNA was extracted using the “QIAamp Fast DNA Stool Mini Kit” from thecompany Qiagen. Shortly, each individual sample from the chicken gut(50-250 mg) was suspended in buffer separating inhibitors from DNA. Thiswas followed by bacterial cell lysis. DNA was then adsorbed to a columnin the presence of chaotropic salts. Washing steps with high-salt liquidand ethanol were used to remove contaminants and DNA was finally elutedusing low-salt or water elution.

PCR Amplification of the 16S RNA Gene

After DNA extraction the extracted DNA was used as template for a PCRreaction targeting the V1-3 variable regions of the 16S rRNA gene.

10-15 ng of extracted DNA was used as template and the PCR reaction (25μL) contained dNTPs (400 nM of each), MgSO4 (1.5 mM), Platinum® Taq DNApolymerase HF (2 mU), 1× Platinum® High Fidelity buffer (Thermo FisherScientific, USA), and barcoded library adaptors (400 nM) containing V3-4specific primers as follows:

Amplification of V1-3 region of 16S RNA gene Forward primer (27F):(SEQ ID NO: 10) AGAGTTTGATCCTGGCTCAG Reverse primer (534R):(SEQ ID NO: 11) ATTACCGCGGCTGCTGG

PCR settings: Initial denaturation at 95° C. for 2 min, 30 cycles of 95°C. for 20 s, 56° C. for 30 s, 72° C. for 60 s and final elongation at72° C. for 5 min. The amplicon libraries were purified using theAgencourt® AMpure XP bead protocol (Beckmann Coulter, USA).

DNA Sequencing

The purified sequencing libraries were pooled and samples were pairedend sequenced (2×301 bp) on a MiSeq (Illumina) using a MiSeq Reagent kitv3, 600 cycles (Illumina) following the standard guidelines forpreparing and loading samples on the MiSeq. 10% Phix control library wasspiked in to overcome low complexity issue often observed with ampliconsamples.

Bioinformatics Processing, OTU Clustering and Classification

Forward and reverse reads were trimmed for quality using Trimmomatic v.0.32 (Bolger, Anthony M., Marc Lohse, and Bjoern Usadel. 2014.“Trimmomatic: A flexible trimmer for Illumina sequence data.”Bioinformatics 30 (15): 2114-20. doi:10.1093/bioinformatics/btu170) withthe settings SLIDINGWINDOW:5:3 and MINLEN:275. The trimmed forward andreverse reads were merged using FLASH v. 1.2.7 (Magoc, Tanja, and StevenL Salzberg. 2011. “FLASH: fast length adjustment of short reads toimprove genome assemblies.” Bioinformatics (Oxford, England) 27 (21):2957-63, doi:10.1093/bioinformatics/btr507) with the settings -m 25-M200. The merged reads were dereplicated and formatted for use in theUPARSE workflow (Edgar, Robert C. 2013. “UPARSE: highly accurate OTUsequences from microbial amplicon reads.” Nature Methods 10 (10): 996-8.doi:10.1038/nmeth.2604). The dereplicated reads were clustered, usingthe usearch v. 7.0.1090-cluster_otus command with default settings. OTUabundances were estimated using the usearch v. 7.0.1090-usearch_globalcommand with -id 0.97. Taxonomy was assigned using the RDP classifier(Wang, Qiong, George M Garrity, James M Tiedje, and James R Cole. 2007.“Naive Bayesian classifierfor rapid assignment of rRNA sequences intothe new bacterial taxonomy.” Applied and Environmental Microbiology 73(16): 5261-7. doi:10.1128/AEM.00062-07.) as implemented in theparallel_assign_taxonomy_rdp.py script in QIIME (Caporaso, J Gregory,Justin Kuczynski, Jesse Stombaugh, Kyle Bittinger, Frederic D Bushman,Elizabeth K Costello, Noah Fierer, et al. 2010. “QIIME allows analysisof high-throughput community sequencing data.” Nature Methods 7 (5),Nature Publishing Group: 335-6. doi:10.1038/nmeth.f.303), using theMiDAS database v.1.20 (Mcllroy, Simon Jon, Aaron Marc Saunders, MadsAlbertsen, Marta Nierychlo, Bianca Mcllroy, Aviaja Anna Hansen, SørenMichael Karst, Jeppe Lund Nielsen, and Per Halkjr Nielsen. 2015. “MiDAS:the field guide to the microbes of activated sludge.” Database 2015 (2):bav062. doi:10.1093/database/bav062).

Statistical Analysis

The results were analysed in R (R Core Team 2015) through the RstudioIDE using the ampvis package v.1.9.1 (Albertsen, Mads, Søren M Karst,Anja S Ziegler, Rasmus H Kirkegaard, and Per H Nielsen. 2015. “Back tobasics—the influence of DNA extraction and primer choice on phylogeneticanalysis of activated sludge communities, PLoS ONE 10(7): e0132783,doi:10.1371/journal.pone.0132783), which builds on the R package DESeq2(Love, Michael I., Wolfgang Huber, and Simon Anders. 2014. “Moderatedestimation of fold change and dispersion for RNA-seq data with DESeq2.”Genome Biology 15 (12): 550. doi:10.1186/s13059-014-0550-8.) fordetecting species in differential abundance and vegan (Oksanen, Jari,Guillaume F Blanchet, Roeland Kindt, Pierre Legendre, Peter R. Minchin,R. B. O'Hara, Gavin L. Simpson, Peter Solymos, Henry H. Stevens, andHelene Wagner. 2015. “vegan: Community Ecology Package”) for ordinationand permutational manova analysis. Pens were used as statistical unitfor the statistical analysis of the microbiota, meaning that theabundances of all bacteria were averaged over all the chickens in eachindividual pen. The detection of species of differential abundancebetween treatment groups was evaluated by p-values adjusted for multipletesting (p_(adj)) such that values of p_(adj) lower than 0.05 wereconsidered significant.

Results

The overall changes in the composition of the chicken gut microbiotaupon treatment with SEQ ID NO: 1 are shown in table 18 below.

TABLE 18 Observed shift in the composition of the microbiota compared tothe control group Trial Lysozyme Concentration Significance¹ FIG. Invivo trial 4 SEQ ID NO: 1 50 ppm +++ FIG. 1 ¹Significant change (+++),p-value < 0.05

A significant shift in the microbial composition in the chicken gut isobserved upon treatment with the lysozyme of SEQ ID NO: 1 and thiseffect is coupled to increased European Production Efficiency Factor(EPEF) in chickens.

The observed changes in the composition of the chicken gut microbiota atoperational taxonomic unit (OTU) level upon treatment with SEQ ID NO: 1are shown in table 19.

TABLE 19 Changes in the chicken gut microbiota at OTU level from in vivobroiler trial 4 Tax. Assignment³ OTU level p-value p_(adj) Control¹Lysozyme² Change Ratio (genus level) OTU #27 0.0010 0.0145 0.22 3.673.45 16.53 Faecalibacterium ¹Treatment 1 (no lysozyme); ²Treatment 2(Lysozyme, SEQ ID NO: 1) ³Taxonomy Assignment

Treatment with SEQ ID NO: 1 leads to a higher proportion of a bacterialspecies of the genus Faecalibacterium in the chicken gut and this shiftis associated with increased European Production Efficiency Factor(EPEF) in chickens and this bacterial species has 96% identity to thespecies Faecalibacterium prausnitzii.

The observed changes in the composition of the chicken gut microbiota atgenus level upon treatment with SEQ ID NO: 1 are shown in table 20below.

TABLE 20 Changes in the chicken gut microbiota at genus level from invivo broiler trial 4 Lyso- Genus level p-value p_(adj) Control¹ zyme²Change Ratio Faecalibacterium 0.123 0.282 1.86 7.50 5.65 4.04 ¹Treatment1 (no lysozyme); ²Treatment 2 (Lysozyme, SEQ ID NO: 1)

Treatment with SEQ ID NO: 1 leads to a higher proportion of bacteria ofthe genus Faecalibacterium in the chicken gut and this shift isassociated with increased European Production Efficiency Factor (EPEF)in chickens.

The observed changes in the composition of the chicken gut microbiota atorder level upon treatment with SEQ ID NO: 1 are shown in table 21below.

TABLE 21 Changes in the chicken gut microbiota at order level from invivo broiler trial 4 Order level p-value p_(adj) Control¹ Lysozyme²Change Ratio Clostridiales 0.0071 0.0570 32.1846 37.5638 5.3792 1.1671Bacteroidales 0.0779 0.2078 64.3989 48.5437 −15.8551 0.7538 ¹Treatment 1(no lysozyme); ²Treatment 2 (Lysozyme, SEQ ID NO: 1)

Treatment with SEQ ID NO: 1 leads to a higher proportion of bacteria ofthe order Clostridiales in the chicken gut and this shift is associatedwith increased European Production Efficiency Factor (EPEF) in chickens.

Treatment with SEQ ID NO: 1 leads to a lower proportion of bacteria ofthe order Bacteroidales in the chicken gut and this shift is associatedwith increased European Production Efficiency Factor (EPEF) in chickens.

A summary of the observed shift in the composition of the microbiotacompared to the control group is presented in table 22 below.

TABLE 22 Observed shift in the composition of the microbiota compared tothe control group Significance¹ FIG. OTU level (Faecalibacieriumspecies) In vivo trial 4 (SEQ ID NO: 1 at 50 ppm) +++ FIG. 2 Genus level(Faecalibacterium) In vivo trial 4 (SEQ ID NO: 1 at 50 ppm) + FIG. 3Order level (Clostridiales) In vivo trial 4 (SEQ ID NO: 1 at 50 ppm) +FIG. 4 Order level (Bacteroidales) In vivo trial 4 (SEQ ID NO: 1 at 50ppm) + FIG. 4 ¹Significant change (+++), p_(adj) < 0.05, Numericalchange (+)

In conclusion it can be seen that the GH25 lysozymes induced asignificant shift in the microbial composition in the chicken gut andthis effect is coupled to an increased European Production EfficiencyFactor (EPEF) in chickens. Treatment with the GH25 lysozyme led to ahigher proportion of bacterial species within the genusFaecalibacterium, and overall increased the proportion of bacteria ofthe order Clostridiales and decreased bacteria of the orderBacteroidales.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A method of improving the European Production Efficiency Factor(EPEF) and/or feed conversion ratio (FCR) of a monogastric animalcomprising administering an animal feed or animal feed additivecomprising one or more microbial lysozymes to the monogastric animalwherein the microbial lysozyme is obtained or obtainable from thekingdom Fungi and is administered at a level of 8 to 250 ppm enzymeprotein per kg animal feed.
 2. The method of claim 1, wherein the FCR isimproved by at least 1.0% compared to control.
 3. The method of claim 1,wherein the EPEF is improved by at least 1.0% compared to control. 4.The method of claim 1, wherein the microbial lysozyme has antimicrobialactivity toward Clostridium perfringens.
 5. The method of claim 1,wherein the animal feed or animal feed additive increases the proportionof bacteria of genus Faecalibacterium in the microbiota of the GI tractof an animal.
 6. The method of claim 5, wherein the proportion ofbacteria of genus Faecalibacterium is increased by at least 1%.
 7. Themethod of claim 5, wherein the proportion of bacteria of genusFaecalibacterium is increased by a factor of at least 1.25.
 8. Themethod of claim 1, wherein the animal feed or animal feed additiveimproves the European Production Efficiency Factor (EPEF) and/or FeedConversion Ratio (FCR) of an animal by at least 1% compared to controland increases the proportion of bacteria of genus Faecalibacterium inthe microbiota of the GI tract of an animal.
 9. The method of claim 8,wherein the proportion of bacteria of genus Faecalibacterium isincreased by at least 1%.
 10. The method of claim 8, wherein theproportion of bacteria of genus Faecalibacterium is increased by afactor of at least 1.25.
 11. The method of claim 1, wherein themonogastric animal is selected from the group consisting of swine,piglet, growing pig, sow, poultry, turkey, duck, quail, guinea fowl,goose, pigeon, squab, chicken, broiler, layer, pullet and chick, horse,crustaceans, shrimps, prawns, fish, amberjack, arapaima, barb, bass,bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla,chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby,goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, leach,mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey,perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream,shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon,sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot,vendace, walleye and whitefish.
 12. The method of claim 1, wherein themonogastric animal is selected from the group consisting of swine,piglet, growing pig, sow, chicken, broiler, layer, pullet and chick. 13.The method of claim 1, wherein the microbial lysozyme is fed to theanimal during the life span of the animal.
 14. The method of claim 1,wherein the microbial lysozyme is fed to broilers during the pre-starter(days 1-7) and/or starter (days 8-22) period or to piglets during thepre-starter (days 1-14 after weaning) and/or starter (days 15-42 afterweaning) period.
 15. The method of claim 1, wherein the microbiallysozyme is obtained or obtainable from the phylum Ascomycota.
 16. Themethod of claim 1, wherein the microbial lysozyme is obtained orobtainable from the subphylum Pezizomycotina.
 17. The method of claim 1,wherein the microbial lysozyme comprises one or more domains selectedfrom the list consisting of GH24 and GH25.
 18. The method of claim 1,wherein the microbial lysozyme is selected from the group consisting of:(a) a polypeptide having at least 50% sequence identity to SEQ ID NO: 1;(b) a variant of SEQ ID NO: 1 wherein the variant has lysozyme activityand comprises one or more amino acid substitutions, and/or one or moreamino acid deletions, and/or one or more amino acid insertions or anycombination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50positions; (c) a fragment of the polypeptide of (a) or (b) that haslysozyme activity wherein the fragment comprises at least 170 aminoacids; (d) a polypeptide having at least 50% sequence identity to SEQ IDNO: 4; (e) a variant of SEQ ID NO: 4 wherein the variant has lysozymeactivity and comprises one or more amino acid substitutions, and/or oneor more amino acid deletions, and/or one or more amino acid insertionsor any combination thereof in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49or 50 positions; and (f) a fragment of the polypeptide of (d) or (e)that has lysozyme activity wherein the fragment comprises at least 210amino acid.
 19. The method of claim 1, wherein the microbial lysozyme isselected from the group consisting of amino acids 1 to 213 of SEQ ID NO:1, amino acids 1 to 245 of SEQ ID NO: 4 and amino acids 1 to 208 of SEQID NO:
 12. 20-25. (canceled)
 26. A method of increasing the proportionof bacteria of genus Faecalibacterium in the microbiome of the GI tractof a monogastric animal comprising administering to the animal an animalfeed or animal feed additive comprising one or more microbial lysozymesadministered at a level of 8 to 250 ppm enzyme protein per kg animalfeed. 27-44. (canceled)